Use of fenoterol and fenoterol analogues in the treatment of glioblastomas and astrocytomas

ABSTRACT

This disclosure concerns the discovery of the use of fenoterol and (R,R)- and (R,S)-fenoterol analogs for the treatment of a tumor expressing a β2-adrenergic receptor, such as a primary brain tumor, including a glioblastoma or astrocytoma expressing a β2-adrenergic receptor. In one example, the method includes administering to a subject a therapeutically effective amount of fenoterol, a specific fenoterol analog or a combination thereof to reduce one or more symptoms associated with the tumor, thereby treating the tumor in the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US2011/027988, filed Mar. 10, 2011, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/312,642, filed Mar. 10, 2010, which isincorporated herein by reference in its entirety.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under contract numberN01-AG-3-1008 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

FIELD

The present disclosure relates to the field of (R,R)-fenoterol and(R,R)- or (R,S)-fenoterol analogues and in particular, to methods oftheir use in treating a tumor that expresses a β2-adrenergic receptor,such as a primary tumor expressing a β2-adrenergic receptor.

BACKGROUND

Cancer is the second leading cause of human death next to coronarydisease in the United States. Worldwide, millions of people die fromcancer every year. In the United States alone, as reported by theAmerican Cancer Society, cancer causes the death of well over ahalf-million people annually, with over 1.2 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise.Cancer is soon predicted to become the leading cause of death.

Brain cancer is particularly difficult to treat because many commontherapeutic agents are not able to pass through the blood brain bather.Further, these tumors are often not detected until they are highlyadvanced. For example, the vast majority of malignant brain tumors aregliomas and astrocytomas, which are extremely lethal as the mediansurvival from diagnoses is 12-15 months. The current clinical approachesto the treatment of gliomas and astrocytomas include a combination ofsurgery, radiation and chemotherapy, but these approaches have notsignificantly improved patient survival. Thus, the development of newtherapies is an important area for drug development.

SUMMARY

This disclosure concerns the discovery that fenoterol and specificfenoterol analogues can be used to treat a tumor that is associated withβ2-adrenergic receptor (AR) expression. The inventors have discoveredthat administration of fenoterol, specific fenoterol analogues orcombinations thereof inhibit one or more signs or symptoms (such astumor growth) associated with a tumor that expresses β2-AR. Using thisdiscovery, the inventors developed the disclosed methods of treating atumor expressing a β2-AR, for example a primary brain tumor expressing aβ2-AR, such as an astrocytoma or glioblastoma expressing a β2-AR.

In some embodiments, the method includes administering a therapeuticallyeffective amount of fenoterol, a fenoterol analogue or a combinationthereof to treat a tumor expressing a β2-AR, such as to reduce orinhibit one or more signs or symptoms associated with the tumor(including inhibiting tumor growth or reducing tumor volume).

Exemplary chemical structures for fenoterol analogues that are highlyeffective at binding β2-ARs and can be used in the disclosed therapiesare provided. By way of example, fenoterol analogues are represented bythe following general formula:

wherein R₁-R₃ independently are hydrogen, acyl, alkoxy carbonyl, aminocarbonyl (carbamoyl) or a combination thereof;

R₄ is H or lower alkyl;

R₅ is lower alkyl,

wherein X, Y¹, Y² and Y³ independently are hydrogen, —OR₆ and —NR₇R₈;

R₆ is independently hydrogen, lower alkyl, acyl, alkoxy carbonyl oramino carbonyl; R₇ and R₈ independently are hydrogen, lower alkyl,alkoxy carbonyl, acyl or amino carbonyl and wherein the compound isoptically active.

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a loweralkyl (such as, CH₃ or CH₂CH₃); R₅ is a lower alkyl,

wherein X, Y¹, Y² and Y³ independently are hydrogen, —OR₆ and —NR₇R₈; R₆is independently hydrogen, lower alkyl, acyl, alkoxy carbonyl or aminocarbonyl; R₇ and R₈ independently are hydrogen, lower alkyl, alkoxycarbonyl, acyl or amino carbonyl and wherein the compound is opticallyactive.

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a methyl oran ethyl; R₅ is

wherein X is —OH or —OCH₃.

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a methyl oran ethyl; R₅ is

In some embodiments, the method includes administering a therapeuticallyeffective amount of a pharmaceutical composition containing fenoterol,any of the disclosed fenoterol analogues or a combination thereof and apharmaceutically acceptable carrier to treat a tumor expressing a β2-AR,such as a primary brain tumor expressing β2-AR expression. For example,the disclosed (R,R)-fenoterol and (R,R)- or (R,S)-fenoterol analogues(e.g., (R,R)-methoxy-ethylfenoterol, (R,R)-methoxyfenoterol,(R,R)-napthylfenoterol, (R,R)-ethylfenoterol and (R,S)-napthylfenoterol)are effective at treating a primary brain tumor expressing a β2-AR, suchas a glioblastoma or astrocytoma expressing β2-AR. In some embodiments,the method further includes selecting a subject having or at risk ofdeveloping a tumor associated with β2-AR expression. For example, asubject is selected for treatment by determining that the tumorexpresses β2-ARs. In one particular example, the method further includesselecting a subject that does not have a bleeding disorder. In furtherexamples, the method includes administering one or more therapeuticagents in addition to fenoterol, a fenoterol analogue or combinationthereof. The methods can include administration of the one or moretherapeutic agents separately, sequentially or concurrently, for examplein a combined composition with fenoterol, a fenoterol analogue orcombinations thereof.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chromatographic separation of (S,S)- and(R,R)-fenoterol.

FIG. 2A is an ultraviolet spectra of (R,R)- and (S,S)-fenoterol.

FIG. 2B illustrates a circular dichroism spectra of (R,R)- and(S,S)-fenoterol.

FIG. 3 provides frontal chromatographic elution profiles of[⁺H]-(±)-fenoterol produced by the addition of (R,R)-fenoterol to therunning buffer.

FIG. 4 illustrates the chemical structures of the stereoisomers offenoterol and fenoterol analogs (compounds 2-7).

FIG. 5 illustrates the chemical structures of compounds 47-51.

FIG. 6 illustrates dose-dependent stimulation of cAMP accumulation byfenoterol analogs. (R,R)-fenoterol (▪) is a full agonist for stimulationof cAMP accumulation in 1321N1 cells, exhibiting greater stimulation ofcAMP than the standard isoproterenol (●) or forskolin (▴).(S,S)-fenoterol (♦) was determined to be a partial agonist in thesecells.

FIGS. 7A and 7B illustrate dose-dependent inhibition of [³H]thymidineincorporation by fenoterol isomers and forskolin. FIG. 7A shows that(R,R)-fenoterol (▪) is 1000 times more potent than both (S,S)-fenoterol(▾) and forskolin (●). FIG. 7B shows the selective β2-AR antagonist ICI118-551 at 1 nM (▾) and 3 nM (♦) induces a parallel rightward shift inthe (R,R)-fenoterol (▪) dose response curve.

FIGS. 8A-8E illustrate (R,R)-fenoterol-modulation of P27 (FIG. 8A),phospho AKT (FIG. 8B), Cyclin D1 (FIG. 8C), Cyclin A (FIG. 8D) andp-Erk1/2 protein expression levels. In each case, the lanes were asfollows: lane 1, control; lane 2, 10⁻¹⁰ M (R,R)-fenoterol; lane 3, 10⁻⁸M(R,R)-fenoterol; and lane 4, 10⁻⁶M (R,R)-fenoterol. Western blots werequantified using densitometry. The relative density of the bands isshown above each western blot.

FIG. 9 illustrates that IV administration of [³H]-(R,R)-methoxyfenoterolis an effective method of delivering [³H]-(R,R)-methoxyfenoterol to thebrain as this compound was demonstrated to pass through the blood brainbather. FIG. 9 provides a comparison of [³H]-(R,R)-methoxyfenoterollevels in brain and plasma isolated from male Sprague-Dawley rats. Thebrain (μg-equiv/g) and plasma (μg-equiv/ml) levels of[³H]-(R,R)-methoxyfenoterol were measured over 60 minutes. Each pointrepresents the mean±s.e. of three rats.

FIG. 10 illustrates (R,R)-methoxyfenoterol-growth inhibition of a 1321N1xenograft implanted in the flank of SKID mice.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Introduction

Fenoterol,5-[1-hydroxy-2[[2-(4-hydroxyphenyl)-1-methylethyl]-amino]ethyl]1,2-benzenediol, is β2-AR agonist that has traditionally been used forthe treatment of pulmonary disorders such as asthma. This drug has twochiral (asymmetric) carbons that can each be independently arranged inan R or S configuration, so that the drug exists in distinct (R,R),(R,S), (S,R) and (S,S) forms known as stereoisomers. Fenoterol iscommercially available as a racemic mixture of the (R,R)- and(S,S)-compounds.

Fenoterol acts as an agonist that binds to and activates the β2-AR. Thisactivity has led to its clinical use in the treatment of asthma becausethis agonist's activity dilates constricted airways. Additionaltherapeutic uses for fenoterol remain to be thoroughly explored.

This disclosure reports the ability of fenoterol, specific fenoterolanalogues or a combination thereof to treat a tumor that expressesβ2-AR. In particular, this disclosure provides fenoterol analogues thatbind the β2-AR with comparable or greater activity than fenoterol. Inone embodiment, the optically active fenoterol analogues aresubstantially purified from a racemic mixture. For example, an opticallyactive fenoterol analogue is purified to represent greater than 90%,often greater than 95% of the composition. These analogues can be usedto treat a tumor that expresses a β2-AR, such as a primary tumorassociated with increased β2-AR expression. It is specificallycontemplated that (R,R)-fenoterol as well as disclosed (R,R)- and(R,S)-fenoterol analogues (or a combination thereof) can be used totreat a primary tumor, such as a primary brain tumor including aglioblastoma or astrocytoma expressing β2-AR.

II. Abbreviations and Terms

AR: adrenergic receptor

CD: circular dichroism

CoMFA: comparative molecular field analysis

HPLC: high performance liquid chromatography

IAM-PC: immobilized artificial membrane chromatographic support

ICYP: [¹²⁵I]cyanopindolol

UV: ultraviolet

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the disclosed subject matter belongs.Definitions of common terms in chemistry terms may be found in TheMcGraw-Hill Dictionary of Chemical Terms, 1985, and The CondensedChemical Dictionary, 1981. As used herein, the singular terms “a,” “an,”and “the” include plural referents unless context clearly indicatesotherwise. Similarly, the word “or” is intended to include “and” unlessthe context clearly indicates otherwise. Also, as used herein, the term“comprises” means “includes.” Hence “comprising A or B” means includingA, B, or A and B.

Except as otherwise noted, any quantitative values are approximatewhether the word “about” or “approximately” or the like are stated ornot. The materials, methods, and examples described herein areillustrative only and not intended to be limiting. Any molecular weightor molecular mass values are approximate and are provided only fordescription. Except as otherwise noted, the methods and techniques ofthe present invention are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See, e.g., Loudon, Organic Chemistry, FourthEdition, New York: Oxford University Press, 2002, pp. 360-361,1084-1085; Smith and March, March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience,2001; or Vogel, A Textbook of Practical Organic Chemistry, IncludingQualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.

In order to facilitate review of the various embodiments disclosedherein, the following explanations of specific terms are provided:

Acyl: A group of the formula RC(O)— wherein R is an organic group.

Acyloxy: A group having the structure —OC(O)R, where R may be anoptionally substituted alkyl or optionally substituted aryl. “Loweracyloxy” groups are those where R contains from 1 to 10 (such as from 1to 6) carbon atoms.

Administration: To provide or give a subject a composition, such as apharmaceutical composition including fenoterol and/or one or morefenoterol analogues, by any effective route. Exemplary routes ofadministration include, but are not limited to, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal (ip), andintravenous (iv)), oral, sublingual, rectal, transdermal, intranasal,vaginal and inhalation routes.

Alkoxy: A radical (or substituent) having the structure —O—R, where R isa substituted or unsubstituted alkyl. Methoxy (—OCH₃) is an exemplaryalkoxy group. In a substituted alkoxy, R is alkyl substituted with anon-interfering substituent. “Thioalkoxy” refers to —S—R, where R issubstituted or unsubstituted alkyl. “Haloalkyloxy” means a radical —ORwhere R is a haloalkyl.

Alkoxy carbonyl: A group of the formula —C(O)OR, where R may be anoptionally substituted alkyl or optionally substituted aryl. “Loweralkoxy carbonyl” groups are those where R contains from 1 to 10 (such asfrom 1 to 6) carbon atoms.

Alkyl: An acyclic, saturated, branched- or straight-chain hydrocarbonradical, which, unless expressly stated otherwise, contains from one tofifteen carbon atoms; for example, from one to ten, from one to six, orfrom one to four carbon atoms. This term includes, for example, groupssuch as methyl, ethyl, n-propyl, isopropyl, isobutyl, t-butyl, pentyl,heptyl, octyl, nonyl, decyl, or dodecyl. The term “lower alkyl” refersto an alkyl group containing from one to ten carbon atoms. Unlessexpressly referred to as an “unsubstituted alkyl,” alkyl groups caneither be unsubstituted or substituted. An alkyl group can besubstituted with one or more substituents (for example, up to twosubstituents for each methylene carbon in an alkyl chain). Exemplaryalkyl substituents include, for instance, amino groups, amide,sulfonamide, halogen, cyano, carboxy, hydroxy, mercapto,trifluoromethyl, alkyl, alkoxy (such as methoxy), alkylthio, thioalkoxy,arylalkyl, heteroaryl, alkylamino, dialkylamino, alkylsulfano, keto, orother functionality.

Amino carbonyl (carbamoyl): A group of the formula C(O)N(R)R′, wherein Rand R′ are independently of each other hydrogen or a lower alkyl group.

Astrocytoma: A tumor of the brain that originates in astrocytes. Anastrocytoma is an example of a primary tumor. Astrocytomas are the mostcommon glioma, and can occur in most parts of the brain and occasionallyin the spinal cord. However, astrocytomas are most commonly found in thecerebrum. In one example, an astrocytoma is inhibited by administering,to a subject, a therapeutic effective amount of fenoterol, a fenoterolanalogue or a combination thereof, thereby inhibiting astrocytomagrowth.

β2-adrenergic receptor (β2-AR): A subtype of adrenergic receptors thatare members of the G-protein coupled receptor family. β2-AR sub-type isinvolved in respiratory diseases, cardiovascular diseases and prematurelabor and as disclosed herein tumor development. Increased expression ofβ2-ARs can serve as therapeutic targets. Currently, a number of drugse.g., albuterol, formoterol, isoproternol, or salmeterol have β2-ARagonist activities. As disclosed herein, fenoterol and fenoterolanalogues are β2-AR agonists. In an example, fenoterol, a fenoterolanalogue or a combination thereof is administered to a subject to reduceor inhibit one or more symptoms or signs associated with a tumorexpressing a β2-AR (such as increased (β2-AR expression), for example aprimary brain tumor expressing a β2-AR.

Blood-brain barrier (BBB): The barrier formed by epithelial cells in thecapillaries that supply the brain and central nervous system. Thisbarrier selectively allows entry of substances such as water, oxygen,carbon dioxide, and nonionic solutes such as glucose, alcohol, andgeneral anesthetics, while blocking entry of other substances. Somesmall molecules, such as amino acids, are taken across the barrier byspecific transport mechanisms. In one example, fenoterol or disclosedfenoterol analogues are capable of passing through the barrier.

Carbamate: A group of the formula —OC(O)N(R)—, wherein R is H, or analiphatic group, such as a lower alkyl group or an aralkyl group.

Chemotherapy; chemotherapeutic agents: As used herein, any chemicalagent with therapeutic usefulness in the treatment of diseasescharacterized by abnormal cell growth. Such diseases include tumors,neoplasms, and cancer as well as diseases characterized by hyperplasticgrowth. In one embodiment, a chemotherapeutic agent is an agent of usein treating neoplasms such as solid tumors, including a tumor associatedwith β2-AR expression. In one embodiment, a chemotherapeutic agent isradioactive molecule. In some embodiments, fenoterol, a fenoterolanalogue or a combination thereof is a chemotherapeutic agent. In oneexample, a chemotherapeutic agent is carmustine, lomustine,procarbazine, streptozocin, or a combination thereof. One of skill inthe art can readily identify a chemotherapeutic agent of use (e.g., seeSlapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison'sPrinciples of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer L., Berkery R. (eds): OncologyPocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995;Fischer D S, Knobf M F, Durivage H J (eds): The Cancer ChemotherapyHandbook, 4th ed. St. Louis, Mosby-Year Book, 1993).

Control or Reference Value: A “control” refers to a sample or standardused for comparison with a test sample. In some embodiments, the controlis a sample obtained from a healthy subject or a tumor tissue sampleobtained from a patient diagnosed with tumor that did not respond totreatment with fenoterol, a fenoterol analogue or a combination thereof.In some embodiments, the control is a historical control or standardreference value or range of values (such as a previously tested controlsample, such as a group of subjects which do not have a tumor expressingβ2-ARs or group of samples that represent baseline or normal values,such as the level of β2-ARs in tumor tissue that does not respond totreatment with fenoterol, a fenoterol analogue or a combinationthereof).

Derivative: A chemical substance that differs from another chemicalsubstance by one or more functional groups. Preferably, a derivative(such as a fenoterol analogue) retains a biological activity (such asβ2-AR stimulation) of a molecule from which it was derived (such as afenoterol or a fenoterol analogue).

Glioblastoma: A common and malignant form of a primary brain tumor. Aglioblastoma is a grade IV astrocytoma and usually spreads rapidly inthe brain. In one example, a glioblastoma is inhibited by administeringa therapeutic effective amount of fenoterol, a fenoterol analogue or acombination thereof to a subject, thereby inhibiting one or moresymptoms associated with the glioblastoma.

Isomers: Compounds that have the same molecular formula but differ inthe nature or sequence of bonding of their atoms or the arrangement oftheir atoms in space are termed “isomers”. Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers”.Stereoisomers that contain two or more chiral centers and are not mirrorimages of one another are termed “diastereomers.” Steroisomers that arenon-superimposable mirror images of each other are termed “enantiomers.”When a compound has an asymmetric center, for example, if a carbon atomis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−) isomers, respectively). A chiralcompound can exist as either an individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

The compounds described herein may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R),(S), (R,R), (R,S)-stereoisomers or as mixtures thereof. Unless indicatedotherwise, the description or naming of a particular compound in thespecification and claims is intended to include both individualenantiomers and mixtures, racemic or otherwise, thereof. The methods forthe determination of stereochemistry and the separation of stereoisomersare well-known in the art (see, e.g., March, Advanced Organic Chemistry,4th edition, New York: John Wiley and Sons, 1992, Chapter 4).

Optional: “Optional” or “optionally” means that the subsequentlydescribed event or circumstance can but need not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more nucleic acidmolecules, proteins or antibodies that bind these proteins, andadditional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Phenyl: Phenyl groups may be unsubstituted or substituted with one, twoor three substituents, with substituent(s) independently selected fromalkyl, heteroalkyl, aliphatic, heteroaliphatic, thioalkoxy, halo,haloalkyl (such as —CF₃), nitro, cyano, —OR (where R is hydrogen oralkyl), —N(R)R′ (where R and R′ are independently of each other hydrogenor alkyl), —COOR (where R is hydrogen or alkyl) or —C(O)N(R′)R″ (whereR′ and R″ are independently selected from hydrogen or alkyl).

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpreparation is one in which a desired component such as an(R,R)-enantiomer of fenoterol is more enriched than it was in apreceding environment such as in a (±)-fenoterol mixture. A desiredcomponent such as (R,R)-enantiomer of fenoterol is considered to bepurified, for example, when at least about 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% of a sample by weight is composed of the desiredcomponent. Purity of a compound may be determined, for example, by highperformance liquid chromatography (HPLC) or other conventional methods.In an example, the specific fenoterol analogue enantiomers are purifiedto represent greater than 90%, often greater than 95% of the otherenantiomers present in a purified preparation. In other cases, thepurified preparation may be essentially homogeneous, wherein otherstereoisomers are less than 1%.

Compounds described herein may be obtained in a purified form orpurified by any of the means known in the art, including silica geland/or alumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. by Snyder and Kirkland, N.Y.: JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed. by Stahl, NewYork: Springer Verlag, 1969. In an example, a compound includes purifiedfenoterol or fenoterol analogue with a purity of at least about 70%,80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weight relativeto other contaminants. In a further example, a compound includes atleast two purified stereoisomers each with a purity of at least about70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weightrelative to other contaminants. For instance, a compound can include asubstantially purified (R,R)-fenoterol analogue and a substantiallypurified (R,S)-fenoterol analogue.

Subject: The term “subject” includes both human and veterinary subjects,for example, humans, non-human primates, dogs, cats, horses, rats, mice,and cows. Similarly, the term mammal includes both human and non-humanmammals.

Therapeutically Effective Amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of (R,R)-fenoterol or(R,R)- or (R,S)-fenoterol analogue useful in reducing, inhibiting,and/or treating a primary tumor, such as a glioblastoma or astrocytomaassociated with β2-AR expression. Ideally, a therapeutically effectiveamount of an agent is an amount sufficient to reduce, inhibit, and/ortreat the disorder in a subject without causing a substantial cytotoxiceffect in the subject.

The effective amount of a composition useful for reducing, inhibiting,and/or treating a disorder in a subject will be dependent on the subjectbeing treated, the severity of the disorder, and the manner ofadministration of the therapeutic composition. Effective amounts atherapeutic agent can be determined in many different ways, such asassaying for a reduction in tumor size or improvement of physiologicalcondition of a subject having a tumor, such as a brain tumor. Effectiveamounts also can be determined through various in vitro, in vivo or insitu assays.

Tissue: A plurality of functionally related cells. A tissue can be asuspension, a semi-solid, or solid. Tissue includes cells collected froma subject such as the brain or a portion thereof.

Tumor: All neoplastic cell growth and proliferation, whether malignantor benign, and all pre-cancerous and cancerous cells and tissues. Aprimary tumor is tumor growing at the anatomical site where tumorprogression began and proceeded to yield this mass. A primary braintumor (also referred to as a glioma) is a tumor that originates in thebrain. Exemplary primary brain tumors include astrocytomas,glioblastomas, ependymoma, oligodendroglomas, and mixed gliomas. In someexamples, a primary brain tumor is associated with β2-AR expression(such as increased β2-AR expression), such as an astrocytoma orglioblastoma associated with β2-AR expression.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity. In one example, underconditions sufficient for includes administering fenoterol and/or one ormore fenoterol analogues or a combination thereof to a subject to at aconcentration sufficient to allow the desired activity. In someexamples, the desired activity is reducing or inhibiting a sign orsymptom associated with a tumor (such as a primary brain tumor) can beevidenced, for example, by a delayed onset of clinical symptoms of thetumor in a susceptible subject, a reduction in severity of some or allclinical symptoms of the tumor, a slower progression of the tumor (forexample by prolonging the life of a subject having the tumor), areduction in the number of tumor reoccurrence, an improvement in theoverall health or well-being of the subject, or by other parameters wellknown in the art that are specific to the particular disease. In oneparticulate example, the desired activity is preventing or inhibitingtumor growth, such as astrocytoma or glioblastoma growth. Tumor growthdoes not need to be completely inhibited for the treatment to beconsidered effective. For example, a partial reduction or slowing ofgrowth such as at least about a 10% reduction, such as at least 20%, atleast about 30%, at least about 40%, at least about 50% or greater isconsidered to be effective.

III. (R,R)-Fenoterol and Fenoterol Analogues

A. Chemical Structure

Some exemplary fenoterol analogues disclosed herein have the formula:

wherein R₁-R₃ independently are hydrogen, acyl, alkoxy carbonyl, aminocarbonyl or a combination thereof;

R₄ is H or lower alkyl;

R₅ is lower alkyl,

wherein X and Y independently are selected from hydrogen, lower —OR₆ and—NR₇R₈;

R₆ is lower alkyl or acyl; and

R₇ and R₈ independently are hydrogen, lower alkyl, alkoxy carbonyl, acylor amino carbonyl.

With continued reference to the general formula for fenoterol analoguesabove, Y may be —OH.

In one embodiment, R₅ is a 1- or 2-napthyl derivative optionally having1, 2 or 3 substituents. Examples of such R₅ groups are represented bythe formula

wherein Y¹, Y² and Y³ independently are hydrogen, lower —OR₆ and —NR₇R₈;

R₆ is independently for each occurrence selected from lower alkyl andacyl; and

R₇ and R₈ independently are hydrogen, lower alkyl, alkoxy carbonyl, acylor amino carbonyl (carbamoyl). In particular compounds at least one ofY¹, Y² and Y³ is —OCH₃.

Particular R₅ groups include those represented by the formulas

wherein R₆ is lower alkyl, such as methyl, ethyl, propyl or isopropyl oracyl, such as acetyl.

Exemplary R₅ groups include

In one example, R₄ is lower alkyl and R₅ is

wherein X and Y independently are selected from H, lower alkyl —OR₆ and—NR₇R₈;

R₆ is lower alkyl; and

R₇ and R₈ independently are hydrogen or lower alkyl.

In a further example, R₄ is selected from ethyl, n-propyl, and isopropyland R₅ has the formula

wherein X is H, —OR₆ or —NR₇R₈. For example, R₆ may be methyl or R₇ andR₈ are hydrogen.

In an additional example, R₅ has the formula

In further embodiments, R₄ is selected from methyl, ethyl, n-propyl andisopropyl and R₅ represents

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a loweralkyl (such as, CH₃ or CH₂CH₃); R₅ is lower alkyl,

wherein X, Y¹, Y² and Y³ independently are hydrogen, —OR₆ and —NR₇R₈; R₆is independently hydrogen, lower alkyl, acyl, alkoxy carbonyl or aminocarbonyl; R₇ and R₈ independently are hydrogen, lower alkyl, alkoxycarbonyl, acyl or amino carbonyl and wherein the compound is opticallyactive.

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a methyl oran ethyl; R₅ is

wherein X is —OH or —OCH₃.

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a methyl oran ethyl; R₅ is

Examples of suitable groups for R₁-R₃ that can be cleaved in vivo toprovide a hydroxy group include, without limitation, acyl, acyloxy andalkoxy carbonyl groups. Compounds having such cleavable groups arereferred to as “prodrugs.” The term “prodrug,” as used herein, means acompound which includes a substituent that is convertible in vivo (e.g.,by hydrolysis) to a hydroxyl group. Various forms of prodrugs are knownin the art, for example, as discussed in Bundgaard, (ed.), Design ofProdrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology,Vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed), Designand Application of Prodrugs, Textbook of Drug Design and Development,Chapter 5, 113 191 (1991); Bundgaard, et al., Journal of Drug DeliveryReviews, 8:1 38 (1992); Bundgaard, Pharmaceutical Sciences, 77:285 etseq. (1988); and Higuchi and Stella (eds.) Prodrugs as Novel DrugDelivery Systems, American Chemical Society (1975).

An exemplary (R,R)-compound has the chemical structure of:

X and R₁-R₃ are as described above.

An additional exemplary (R,R)-compound has the chemical structure:

An exemplary (R,S)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

An additional exemplary (R,S)-compound has the chemical structure:

An exemplary (S,R)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

An exemplary (S,S)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

Examples of chemical structures illustrating the various stereoisomersof fenoterol are provided below.

Particular method embodiments contemplate the use of solvates (such ashydrates), pharmaceutically acceptable salts and/or different physicalforms of (R,R)-fenoterol or any of the fenoterol analogues hereindescribed.

1. Solvates, Salts and Physical Forms

“Solvate” means a physical association of a compound with one or moresolvent molecules. This physical association involves varying degrees ofionic and covalent bonding, including by way of example covalent adductsand hydrogen bonded solvates. In certain instances the solvate will becapable of isolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. “Solvate”encompasses both solution-phase and isolable solvates. Representativesolvates include ethanol associated compound, methanol associatedcompounds, and the like. “Hydrate” is a solvate wherein the solventmolecule(s) is/are H₂O.

The disclosed compounds also encompass salts including, if severalsalt-forming groups are present, mixed salts and/or internal salts. Thesalts are generally pharmaceutically-acceptable salts that arenon-toxic. Salts may be of any type (both organic and inorganic), suchas fumarates, hydrobromides, hydrochlorides, sulfates and phosphates. Inan example, salts include non-metals (e.g., halogens) that form groupVII in the periodic table of elements. For example, compounds may beprovided as a hydrobromide salt.

Additional examples of salt-forming groups include, but are not limitedto, a carboxyl group, a phosphonic acid group or a boronic acid group,that can form salts with suitable bases. These salts can include, forexample, nontoxic metal cations which are derived from metals of groupsIA, IB, IIA and IIB of the periodic table of the elements. In oneembodiment, alkali metal cations such as lithium, sodium or potassiumions, or alkaline earth metal cations such as magnesium or calcium ionscan be used. The salt can also be a zinc or an ammonium cation. The saltcan also be formed with suitable organic amines, such as unsubstitutedor hydroxyl-substituted mono-, di- or tri-alkylamines, in particularmono-, di- or tri-alkylamines, or with quaternary ammonium compounds,for example with N-methyl-N-ethylamine, diethylamine, triethylamine,mono-, bis- or tris-(2-hydroxy-lower alkyl)amines, such as mono-, bis-or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine ortris(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-loweralkyl)amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine, or N-methyl-D-glucamine, or quaternaryammonium compounds such as tetrabutylammonium salts.

Exemplary compounds disclosed herein possess at least one basic groupthat can form acid-base salts with inorganic acids. Examples of basicgroups include, but are not limited to, an amino group or imino group.Examples of inorganic acids that can form salts with such basic groupsinclude, but are not limited to, mineral acids such as hydrochloricacid, hydrobromic acid, sulfuric acid or phosphoric acid. Basic groupsalso can form salts with organic carboxylic acids, sulfonic acids, sulfoacids or phospho acids or N-substituted sulfamic acid, for exampleacetic acid, propionic acid, glycolic acid, succinic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid, and, in addition,with amino acids, for example with α-amino acids, and also withmethanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonicacid, ethane-1,2-disulfonic acid, benzenedisulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid(with formation of the cyclamates) or with other acidic organiccompounds, such as ascorbic acid. In a currently preferred embodiment,fenoterol is provided as a hydrobromide salt and exemplary fenoterolanalogues are provided as their fumarate salts.

Additional counterions for forming pharmaceutically acceptable salts arefound in Remington's Pharmaceutical Sciences, 19th Edition, MackPublishing Company, Easton, Pa., 1995. In one aspect, employing apharmaceutically acceptable salt may also serve to adjust the osmoticpressure of a composition.

In certain embodiments the compounds used in the method are provided arepolymorphous. As such, the compounds can be provided in two or morephysical forms, such as different crystal forms, crystalline, liquidcrystalline or non-crystalline (amorphous) forms.

2. Use for the Manufacture of a Medicament

Any of the above described compounds (e.g., (R,R)-fenoterol or fenoterolanalogues or a hydrate or pharmaceutically acceptable salt thereof) orcombinations thereof are intended for use in the manufacture of amedicament for β2-AR stimulation in a subject or for the treatment of aprimary brain tumor (e.g., astrocytoma or glioblastoma). Formulationssuitable for such medicaments, subjects who may benefit from same andother related features are described elsewhere herein.

B. Methods of Synthesis

The disclosed fenoterol analogues can be synthesized by any method knownin the art. Many general references providing commonly known chemicalsynthetic schemes and conditions useful for synthesizing the disclosedcompounds are available (see, e.g., Smith and March, March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein may be purified by any of the means knownin the art, including chromatographic means, such as HPLC, preparativethin layer chromatography, flash column chromatography and ion exchangechromatography. Any suitable stationary phase can be used, includingnormal and reversed phases as well as ionic resins. Most typically thedisclosed compounds are purified via open column chromatography or prepchromatography.

Suitable exemplary syntheses of fenoterol analogues are provided below:

IV. Pharmaceutical Compositions

The disclosed (R,R)-fenoterol and fenoterol analogues can be useful, atleast, for the treatment of a tumor that expresses a β2-AR, (such asincreased β2-AR expression), including a primary brain tumor (e.g., anastrocytoma or glioblastoma) expressing β2-AR. Accordingly,pharmaceutical compositions comprising at least one disclosed fenoterolcompound and/or analogue are also described herein.

Formulations for pharmaceutical compositions are well known in the art.For example, Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition, 1995, describes exemplaryformulations (and components thereof) suitable for pharmaceuticaldelivery of (R,R)-fenoterol and disclosed fenoterol analogues.Pharmaceutical compositions comprising at least one of these compoundscan be formulated for use in human or veterinary medicine. Particularformulations of a disclosed pharmaceutical composition may depend, forexample, on the mode of administration (e.g., oral or parenteral) and/oron the disorder to be treated (e.g., a tumor associated with β2-ARexpression, such as a primary brain tumor). In some embodiments,formulations include a pharmaceutically acceptable carrier in additionto at least one active ingredient, such as a fenoterol compound.

Pharmaceutically acceptable carriers useful for the disclosed methodsand compositions are conventional in the art. The nature of apharmaceutical carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions such as powder, pill, tablet, or capsuleforms conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can optionally contain minor amounts ofnon-toxic auxiliary substances or excipients, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like;for example, sodium acetate or sorbitan monolaurate. Other non-limitingexcipients include, nonionic solubilizers, such as cremophor, orproteins, such as human serum albumin or plasma preparations.

The disclosed pharmaceutical compositions may be formulated as apharmaceutically acceptable salt. Pharmaceutically acceptable salts arenon-toxic salts of a free base form of a compound that possesses thedesired pharmacological activity of the free base. These salts may bederived from inorganic or organic acids. Non-limiting examples ofsuitable inorganic acids are hydrochloric acid, nitric acid, hydrobromicacid, sulfuric acid, hydriodic acid, and phosphoric acid. Non-limitingexamples of suitable organic acids are acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, methyl sulfonic acid,salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid,gluconic acid, asparagic acid, aspartic acid, benzenesulfonic acid,p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. Lists ofother suitable pharmaceutically acceptable salts are found inRemington's Pharmaceutical Sciences, 19th Edition, Mack PublishingCompany, Easton, Pa., 1995. A pharmaceutically acceptable salt may alsoserve to adjust the osmotic pressure of the composition.

The dosage form of a disclosed pharmaceutical composition will bedetermined by the mode of administration chosen. For example, inaddition to injectable fluids, oral dosage forms may be employed. Oralformulations may be liquid such as syrups, solutions or suspensions orsolid such as powders, pills, tablets, or capsules. Methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthe art.

Certain embodiments of the pharmaceutical compositions comprising adisclosed compound may be formulated in unit dosage form suitable forindividual administration of precise dosages. The amount of activeingredient such as (R,R)-fenoterol administered will depend on thesubject being treated, the severity of the disorder, and the manner ofadministration, and is known to those skilled in the art. Within thesebounds, the formulation to be administered will contain a quantity ofthe extracts or compounds disclosed herein in an amount effective toachieve the desired effect in the subject being treated.

In particular examples, for oral administration the compositions areprovided in the form of a tablet containing from about 1.0 to about 50mg of the active ingredient, particularly about 2.0 mg, about 2.5 mg, 5mg, about 10 mg, or about 50 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 1 mgto about 50 mg (such as about 2 mg to about 10 mg) active ingredient isadministered two to four times a day, such as two times, three times orfour times.

In other examples, a suitable dose for parental administration is about1 milligram per kilogram (mg/kg) to about 100 mg/kg, such as a dose ofabout 10 mg/kg to about 80 mg/kg, such including about 1 mg/kg, about 2mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg,about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administeredparenterally. However, other higher or lower dosages also could be used,such as from about 0.001 mg/kg to about 1 g/kg, such as about 0.1 toabout 500 mg/kg, including about 0.5 mg/kg to about 200 mg/kg.

Single or multiple administrations of the composition comprising one ormore of the disclosed compositions can be carried out with dose levelsand pattern being selected by the treating physician. Generally,multiple doses are administered. In a particular example, thecomposition is administered parenterally once per day. However, thecomposition can be administered twice per day, three times per day, fourtimes per day, six times per day, every other day, twice a week, weekly,or monthly. Treatment will typically continue for at least a month, moreoften for two or three months, sometimes for six months or a year, andmay even continue indefinitely, i.e., chronically. Repeat courses oftreatment are also possible.

In one embodiment, the pharmaceutical composition is administeredwithout concurrent administration of a second agent for the treatment ofa tumor that expresses a β2-AR. In one specific, non-limiting example,one or more of the disclosed compositions is administered withoutconcurrent administration of other agents, such as without concurrentadministration of an additional agent also known to target the tumor. Inother specific non-limiting examples, a therapeutically effective amountof a disclosed pharmaceutical composition is administered concurrentlywith an additional agent, including an additional therapy (such as, butnot limited to, a chemotherapeutic agent, an additional β2-AR agonist,an anti-inflammatory agent, an anti-oxidant, or other agents known tothose of skill in the art). For example, the disclosed compounds areadministered in combination with a chemotherapeutic agent,anti-oxidants, anti-inflammatory drugs or combinations thereof.

In other examples, a disclosed pharmaceutical composition isadministered an adjuvant therapy. For example, a pharmaceuticalcomposition containing one or more of the disclosed compounds isadministered orally daily to a subject in order to prevent or retardtumor growth. In one particular example, a composition containing equalportions of two or more disclosed compounds is provided to a subject. Inone example, a composition containing unequal portions of two or moredisclosed compounds is provided to the subject. For example, acomposition contains unequal portions of a (R,R)-fenoterol derivativeand a (S,R)-fenoterol derivative and/or a (R,S)-derivative. In oneparticular example, the composition includes a greater amount of the(S,R)- or (R,S)-fenoterol derivative (such as about 3 parts(S,R)-methoxy-ethylfenoterol or 3 parts (S,R)-methyloxy-fenoterol) thanan (R,R)-fenoterol derivative (such as 1 part(R,R)-methoxy-ethylfenoterol or 1 part (R,R)-methyloxy-fenoterol). Suchtherapy can be given to a subject for an indefinite period of time toinhibit, prevent or reduce tumor reoccurrence.

V. Methods of Use

The present disclosure includes methods of treating disorders includingreducing or inhibiting one or more signs or symptoms associated with atumor expressing a β2-AR, such as a primary tumor. In some examples, theprimary tumor is a primary brain tumor expressing a β2-AR, such as anincrease in β2-AR expression. In one example, the primary brain tumorexpressing β2-AR is an astrocytoma. In other examples, the primary braintumor expressing β2-AR is a glioblastoma.

Disclosed methods include administering fenoterol, such as(R,R)-fenoterol, a disclosed fenoterol analogue or a combination thereof(and, optionally, one or more other pharmaceutical agents) to a subjectin a pharmaceutically acceptable carrier and in an amount effective totreat the tumor expressing a β2-AR, such as a primary tumor. Treatmentof a tumor includes preventing or reducing signs or symptoms associatedwith the presence of such tumor (for example, by reducing the size orvolume of the tumor or a metastasis thereof). Such reduced growth can insome examples decrease or slow metastasis of the tumor, or reduce thesize or volume of the tumor by at least 10%, at least 20%, at least 50%,or at least 75%, such as between 10%-90%, 20%-80%, 30%-70%, 40%-60%,including a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 90%, or 95% reduction. In another example, treatmentincludes reducing the invasive activity of the tumor in the subject, forexample by reducing the ability of the tumor to metastasize. In someexamples, treatment using the methods disclosed herein prolongs the timeof survival of the subject.

Routes of administration useful in the disclosed methods include but arenot limited to oral and parenteral routes, such as intravenous (iv),intraperitoneal (ip), rectal, topical, ophthalmic, nasal, andtransdermal as described in detail above.

An effective amount of fenoterol, such as (R,R)-fenoterol, or adisclosed fenoterol analogue or combination thereof will depend, atleast, on the particular method of use, the subject being treated, theseverity of the tumor, and the manner of administration of thetherapeutic composition. A “therapeutically effective amount” of acomposition is a quantity of a specified compound sufficient to achievea desired effect in a subject being treated. For example, this may bethe amount of (R,R)-fenoterol, a disclosed fenoterol analogue or acombination thereof necessary to prevent or inhibit primary brain tumorgrowth and/or one or more symptoms associated with the primary braintumor in a subject. Ideally, a therapeutically effective amount of(R,R)-fenoterol or a disclosed fenoterol analogue is an amountsufficient to prevent or inhibit a tumor, such as a primary brain tumorgrowth and/or one or more symptoms associated with the tumor in asubject without causing a substantial cytotoxic effect on host cells.

Therapeutically effective doses of a disclosed fenoterol compound orpharmaceutical composition can be determined by one of skill in the art,with a goal of achieving concentrations that are at least as high as theIC₅₀ of the applicable compound disclosed in the examples herein. Anexample of a dosage range is from about 0.001 to about 10 mg/kg bodyweight orally in single or divided doses. In particular examples, adosage range is from about 0.005 to about 5 mg/kg body weight orally insingle or divided doses (assuming an average body weight ofapproximately 70 kg; values adjusted accordingly for persons weighingmore or less than average). For oral administration, the compositionsare, for example, provided in the form of a tablet containing from about1.0 to about 50 mg of the active ingredient, particularly about 2.5 mg,about 5 mg, about 10 mg, or about 50 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 1 mgto about 50 mg active ingredient is administered two to four times aday, such as two times, three times or four times.

In other examples, a suitable dose for parental administration is about1 milligram per kilogram (mg/kg) to about 100 mg/kg, such as a dose ofabout 10 mg/kg to about 80 mg/kg, such including about 1 mg/kg, about 2mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg,about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administeredparenterally. However, other higher or lower dosages also could be used,such as from about 0.001 mg/kg to about 1 g/kg, such as about 0.1 toabout 500 mg/kg, including about 0.5 mg/kg to about 200 mg/kg.

Single or multiple administrations of the composition comprising one ormore of the disclosed compositions can be carried out with dose levelsand pattern being selected by the treating physician. Generally,multiple doses are administered. In a particular example, thecomposition is administered parenterally once per day. However, thecomposition can be administered twice per day, three times per day, fourtimes per day, six times per day, every other day, twice a week, weekly,or monthly. Treatment will typically continue for at least a month, moreoften for two or three months, sometimes for six months or a year, andmay even continue indefinitely, i.e., chronically. Repeat courses oftreatment are also possible.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex and diet of the subject, mode and time of administration,rate of excretion, drug combination, and severity of the condition ofthe subject undergoing therapy.

Selecting a Subject

Subjects can be screened prior to initiating the disclosed therapies,for example to select a subject in need of tumor inhibition, such as asubject having or at risk of developing a tumor that expresses β2-AR.Briefly, the method can include screening subjects to determine if theyare in need of tumor inhibition. Subjects having a tumor that expressesa β2-AR, such as a primary tumor, including a primary brain tumor or atrisk of developing such tumor are selected. In one example, subjects arediagnosed with the tumor by clinical signs, laboratory tests, or both.For example, a tumor, such as a primary brain tumor can be diagnosed bycharacteristic clinical signs, such as headaches, vomiting, seizures,dizziness, weight loss and various associated complaints. Diagnosis isgenerally by imaging analysis such as by magnetic resonance imaging(MRI) and confirmed by histology. In some examples, a subject isselected that does not have a bleeding disorder, such as anintracerebral hemorrhage.

In an example, a subject in need of the disclosed therapies is selectedby detecting a tumor expressing a β2-AR, such as by detecting β2-ARexpression in a sample obtained from a subject identified as having,suspected of having or at risk of acquiring such a tumor. For example,detection of an increase, such as at least a 10% increase, including a10%-90%, 20%-80%, 30%-70%, 40%-60%, such as a 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% increase ormore in β2-AR expression as compared to β2-AR expression in the absenceof a primary tumor, indicates that the tumor can be treated using thecompositions and methods provided herein. In other examples, a subjectis selected by detecting a primary brain tumor such as an astrocytoma orglioblastoma by MRI or positron emission tomography (PET) in a subject.

Pre-screening is not required prior to administration of the therapeuticagents disclosed herein (such as those including fenoterol, a fenoterolanalogue or a combination thereof).

Exemplary Tumors

Exemplary tumors include tumors which may express β2-AR includingprimary tumors, such as a primary brain tumor. A primary brain tumorincludes astrocytomas, glioblastomas, ependymoma, oligodendroglomas, andmixed gliomas. Additional possible types of tumors associated with β2-ARexpression include hematological tumors, such as leukemias, includingacute leukemias (such as 11q23-positive acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, acute myelogenousleukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (indolent and high grade forms), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of possible solid tumors which may express β2-AR, includesarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer(including basal breast carcinoma, ductal carcinoma and lobular breastcarcinoma), lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as aglioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma and retinoblastoma). In severalexamples, a tumor is a brain cancer, breast cancer or multiple myelomathat expresses a β2-AR. Tumors expressing β2-ARs can be identified byroutine methods known to those of skill in the art including Westernblot and histological studies with antibodies capable of detectingβ2-ARs.

Assessment

Following the administration of one or more therapies, subjects having atumor expressing β2-AR (for example, a primary tumor) can be monitoredfor decreases in tumor growth, tumor volume or in one or more clinicalsymptoms associated with the tumor. In particular examples, subjects areanalyzed one or more times, starting 7 days following treatment.Subjects can be monitored using any method known in the art includingthose described herein including imaging analysis.

Additional Treatments and Additional Therapeutic Agents

In particular examples, if subjects are stable or have a minor, mixed orpartial response to treatment, they can be re-treated afterre-evaluation with the same schedule and preparation of agents that theypreviously received for the desired amount of time, including theduration of a subject's lifetime. A partial response is a reduction,such as at least a 10%, at least a 20%, at least a 30%, at least a 40%,at least a 50%, or at least a 70% reduction in one or more signs orsymptoms associated with a tumor, such as primary brain tumor, includingtumor size or volume.

In some examples, the method further includes administering atherapeutic effective amount of fenoterol, a fenoterol analogue or acombination thereof with additional therapeutic treatments. Inparticular examples, prior to, during, or following administration of atherapeutic amount of an agent that prevents or inhibits a tumorexpressing a β2-AR, the subject can receive one or more other therapies.In one example, the subject receives one or more treatments to remove orreduce the tumor prior to administration of a therapeutic amount of acomposition including fenoterol, a fenoterol analogue or combinationthereof.

Examples of such therapies include, but are not limited to, surgicaltreatment for removal or reduction of the tumor (such as surgicalresection, cryotherapy, or chemoembolization), as well as anti-tumorpharmaceutical treatments which can include radiotherapeutic agents,anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agentsand antioxidants, kinase inhibitors, and other agents. Particularexamples of additional therapeutic agents that can be used includemicrotubule binding agents, DNA intercalators or cross-linkers, DNAsynthesis inhibitors, DNA and/or RNA transcription inhibitors,antibodies, enzymes, enzyme inhibitors, and gene regulators. Theseagents (which are administered at a therapeutically effective amount)and treatments can be used alone or in combination. Methods andtherapeutic dosages of such agents are known to those skilled in theart, and can be determined by a skilled clinician.

“Microtubule binding agent” refers to an agent that interacts withtubulin to stabilize or destabilize microtubule formation therebyinhibiting cell division. Examples of microtubule binding agents thatcan be used in conjunction with the disclosed therapy include, withoutlimitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine(navelbine), the epothilones, colchicine, dolastatin 15, nocodazole,podophyllotoxin and rhizoxin. Analogs and derivatives of such compoundsalso can be used and are known to those of ordinary skill in the art.For example, suitable epothilones and epothilone analogs are describedin International Publication No. WO 2004/018478. Taxoids, such aspaclitaxel and docetaxel, as well as the analogs of paclitaxel taught byU.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.

The following classes of compounds are of use in the methods disclosedherein: Suitable DNA and/or RNA transcription regulators, including,without limitation, actinomycin D, daunorubicin, doxorubicin andderivatives and analogs thereof also are suitable for use in combinationwith the disclosed therapies. DNA intercalators and cross-linking agentsthat can be administered to a subject include, without limitation,cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C,bleomycin, chlorambucil, cyclophosphamide and derivatives and analogsthereof. DNA synthesis inhibitors suitable for use as therapeutic agentsinclude, without limitation, methotrexate, 5-fluoro-5′-deoxyuridine,5-fluorouracil and analogs thereof. Examples of suitable enzymeinhibitors include, without limitation, camptothecin, etoposide,formestane, trichostatin and derivatives and analogs thereof. Examplesof alkylating agents include carmustine or lomustine. Suitable compoundsthat affect gene regulation include agents that result in increased ordecreased expression of one or more genes, such as raloxifene,5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen, 4-hydroxytamoxifen,mifepristone and derivatives and analogs thereof. Kinase inhibitorsinclude Gleevac, Iressa, and Tarceva that prevent phosphorylation andactivation of growth factors.

Other therapeutic agents, for example anti-tumor agents, that may or maynot fall under one or more of the classifications above, also aresuitable for administration in combination with the disclosed therapies.By way of example, such agents include adriamycin, apigenin, rapamycin,zebularine, cimetidine, and derivatives and analogues thereof.

In one example, at least a portion of the tumor (such as the primarybrain tumor) is surgically removed (for example via cryotherapy),irradiated, chemically treated (for example via chemoembolization) orcombinations thereof, prior to administration of the disclosed therapies(such as administration of fenoterol, a fenoterol analogue or acombination thereof). For example, a subject having a primary braintumor associated with β2-AR expression can have at least a portion ofthe tumor surgically excised prior to administration of the disclosedtherapies. In an example, one or more chemotherapeutic agents areadministered following treatment with a composition including fenoterol,a fenoterol analogue or a combination thereof. In another particularexample, the subject has a primary brain tumor and is administeredradiation therapy, chemoembolization therapy, or both concurrently withthe administration of the disclosed therapies.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

EXAMPLES Example 1 Materials and Methods

Reagents.

Phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin, pepstatinA, MgCl₂, EDTA, Trizma-Hydrochloride (Tris-HCl), (±)-propranolol andminimal essential medium (MEM) were obtained from Sigma Aldrich (St.Louis, Mo.). Egg phosphatidylcholine lipids (PC) were obtained fromAvanti Polar Lipids (Alabaster, Ala.). (±)-fenoterol was purchased fromSigma Aldrich and [³H]-(±)-fenoterol was acquired from AmershamBiosciences (Boston, Mass.). The organic solvents n-hexane, 2-propanoland triethylamine were obtained as ultra pure HPLC grade solvents fromCarlo Erba (Milan, Italy). Fetal bovine serum andpenicillin-streptomycin were purchased from Life Technologies(Gaithersburg, Md.), and [¹²⁵I]-(±)-iodocyanopindolol (ICYP) waspurchased from NEN Life Science Products, Inc. (Boston, Mass.).

Preparation and Identification of (R,R)-Fenoterol and (S,S)-Fenoterol.

(R,R)-fenoterol and (S,S)-fenoterol were prepared from (±)-fenoterolusing chiral HPLC techniques employing an HPLC column (25 cm×0.46 cmi.d.) containing the amylose tris-(3,5-dimethylphenylcarbamate) chiralstationary phase (CHIRALPAK® AD CSP, Chiral Technologies, West Chester,Pa.; CHIRALPAK is a registered trademark of Daicel Chemical IndustriesLtd., Exton, Pa.). The chromatographic system consisted of a JASCO®PU-980 solvent delivery system, and a JASCO® MD-910 multi-wavelengthdetector set at λ=230 nm, connected to a computer workstation; JASCO isa registered trademark of JASCO, Inc., Tokyo, Japan). A Rheodyne model7125 injector with 20 μl sample loop was used to inject 0.2-0.3 mg(±)-fenoterol onto the chromatographic system. The mobile phase wasn-hexane/2-propanol (88/12 v/v) with 0.1% triethylamine, the flow ratewas 1 mL/minute and the temperature of the system was maintained at 25°C. using a column heater/chiller (Model 7955, Jones Chromatography Ltd.,UK). The separated (R,R)-fenoterol and (S,S)-fenoterol were collected in10-mL fractions as the respective peaks eluted from the chromatographiccolumn. A 2-mL intermediate fraction was collected and discarded toimprove the enantiomeric purity of the collected isomers.

The stereochemical configurations of the resolved (R,R)-fenoterol and(S,S)-fenoterol were established using circular dichroism (CD)measurements obtained with a JASCO® J-800 spectropolarimeter. The(R,R)-fenoterol and (S,S)-fenoterol were dissolved in 2-propanol and themeasurements were obtained using 1 cm path length at room temperature.

Immobilized β2-AR Frontal Chromatography.

The liquid chromatography column containing the immobilized β2-AR wasprepared using a previously described technique (Beigi et al., Anal.Chem., 76: 7187-7193, 2004). In brief, cellular membranes were obtainedfrom a HEK 293 cell line that had been transfected with cDNA encodinghuman β2-AR. An aliquot of a cell pellet suspension corresponding to 5-7mg total protein, as determined by the micro BCA method, was used tocreate the column. The membranes were prepared in 10 mL buffer composedof Tris-HCl [50 mM, pH 7.4] containing MgCl₂ (2 mM), benzamidine (1 mM),leupeptin (0.03 mM) pepstatin A (0.005 mM) and EDTA (1 mM).

A 180 mg aliquot of immobilized artificial membrane chromatographicsupport (IAM-PC, 12 micron particle size, 300 Åpore size obtained fromRegis Chemical Co., Morton Grove, Ill.) and 80 μM PC were added to themembrane preparation and the resulting mixture was stirred at roomtemperature for 3 hours, transferred into (5 cm length) nitrocellulosedialysis membrane (MW cutoff 10,000 Da, Pierce Chemical, Rockford, Ill.)and placed in 1 L of dialysis buffer composed of Tris-HCl [50 mM, pH7.4] containing EDTA (1 mM), MgCl₂ (2 mM), NaCl (300 mM) and PMSF (0.2mM) at 4° C. for 24 hours. The dialysis step was repeated twice usingfresh buffer.

After dialysis, the mixture was centrifuged at 120×g for 3 minutes, thesupernatant was discarded and the pellet of IAM support containing theimmobilized receptor-bearing membranes was collected. The pellet wasresuspended in 2 mL chromatographic running buffer, composed of Tris-HCl[10 mM, pH 7.4] containing EDTA (1 mM) and MgCl₂ (2 mM) and thesuspension was pumped through a HR 5/2 chromatographic glass column(Amersham Pharmacia Biotech, Uppsala, Sweden) at a flow rate of 0.3mL/minutes using a peristaltic pump. The end adaptors were assembledproducing a total gel-bed volume of 0.4 mL. The column was stored at 4°C. when not in use.

The column containing the immobilized β2-AR stationary phase was placedin a chromatographic system composed of a HPLC pump (10-AD, ShimadzuInc., Columbia, Md.), a manually controlled FPLC injector (AmershamBiotechnology, Uppsala, Sweden) with a 50 μL sample loop, the packedimmobilized receptor column and an on-line radioactive flow detector(IN/US, Tampa, Fla.), all connected sequentially. In the frontalchromatographic studies, sample volumes of 5-7 mL were appliedcontinuously until the elution profile showed a plateau region. Therunning buffer was composed of Tris-HCl [10 mM, pH 7.4] containing EDTA(1 mM) and MgCl₂ (2 mM) and 0.05 nM [³H]-(±)-fenoterol, the markerligand. (R,R)-fenoterol or (S,S)-fenoterol was added to the runningbuffer in sequential concentrations of 0.1, 80.0, 240, and 700 nM, andapplied to the column. The immobilized receptor column was equilibratedwith about 80 mL of running buffer, without the added (R,R)-fenoterol or(S,S)-fenoterol respectively, in between each sample injection. Allchromatographic studies were carried out at room temperature at a flowrate of 0.2 mL/minutes.

The data were analyzed to determine the number of binding sites anddissociation constant using the non-linear equation (1),

$\begin{matrix}{{\lbrack M\rbrack\left( {{Vi} - {V\;\min}} \right)} = \frac{P\lbrack M\rbrack}{{Kd} + \lbrack M\rbrack}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$where V₁ is the solute elution volume, V_(min) is the elution volume atthe saturation point, P is the number of available binding sites, M isthe concentration of the marker ligand and K_(d) is the dissociationconstant of the ligand.

Ligand-Displacement Binding.

Twenty-four hours after adenoviral infection with human β2-AR, HEK293cells were harvested in lysis buffer, Tris-HCl [5 mM, pH 7.4] containingEGTA [5 mM], and homogenized with 15 strokes on ice. Samples werecentrifuged at 30,000×g for 15 minutes to pellet membranes. Membraneswere resuspended in binding buffer, Tris-HCl [20 mM, pH 7.4] containingNaCl (120 mM), KCl (5.4 mM), CaCl₂ (1.8 mM), MgCl₂ (0.8 mM), and glucose(5 mM) and stored in aliquots at −80° C. Binding assays were performedon 5-10 μg of membrane protein using saturating amounts (1-300 μM) ofthe β-AR-specific ligand [¹²⁵I]cyanopindolol (ICYP). For competitionbinding, the 5-10 μg of membrane protein were pretreated with 50 μM ofGTP_(γs) (non-hydrolyzable guanosine triphosphate) and then incubatedwith ¹²⁵ICYP (50 μM) and different concentrations of fenoterol or itsisomers in a total volume of 250 μL. Nonspecific binding was determinedin the presence of 20 μM propranolol. Reactions were conducted in 250 μLof binding buffer at 37° C. for 1 hour. The binding reaction wasterminated by addition of ice-cold Tris-HCl [10 mM, pH 7.4] to themembrane suspension, followed by rapid vacuum filtration throughglass-fiber filters (Whatman GF/C). Each filter was washed three timeswith an additional 7 mL of ice-cold Tris-HCl [10 mM, pH 7.4]. Theradioactivity of the wet filters was determined in a gamma counter. Allassays were performed in duplicate, and receptor density was normalizedto milligrams of membrane protein. K_(d) and the maximal number ofbinding sites (B_(max)) for ICYP were determined by Scatchard analysisof saturation binding isotherms. Data from competition studies wereanalyzed using 1- or 2-site competition binding curves with GRAPHPADPRISM® Software (GRAPHPAD PRISM is a registered trademark of GraphPadSoftware, Inc., San Diego, Calif.).

Example 2 Purification and Identification of (R,R)-Fenoterol and(S,S)-Fenoterol

This example demonstrates the resolution of (R,R)-fenoterol and(S,S)-fenoterol from (±)-fenoterol to a high degree of enantiomericpurity.

Using the chromatographic conditions described in Example 1,(±)-fenoterol was separated into its component enantiomers,(R,R)-fenoterol and (S,S)-fenoterol, on the AD-CSP. As illustrated inFIG. 1, the two stereoisomers were resolved with enantioselectivityfactor (α) of 1.21 and a resolution factor (R_(S)) of 1.06. Because ofthe observed tailing of the chromatographic peaks, a 2-mL intermediatefraction was collected and discarded. The collected peaks were analyzedusing the same chromatographic conditions and the data demonstrated thatboth enantiomers had been prepared with >97% stereochemical purity.

The assignment of the absolute configuration of the isolated fractionswas accomplished using their chiroptical properties. The ultraviolet(UV) spectra of both fractions contained identical maxima at about 280and 230, indicating the same UV chromophores for the two enantiomers.The circular dichroism (CD) spectrum shows, for the less retainedenantiomeric fraction, negative CD bands at about 280 and 215 nm, whilethe spectrum is positive at 230 and 200 nm. The sign of the CD bands isreversed for the most retained fenoterol fraction, this confirming theenantiomeric nature of the two fractions. The lowest energy UV and CDspectra of the two enantiomeric fractions are presented in FIGS. 2A and2B, respectively. The less retained chromatographic fraction showed anegative CD band at about 280 nm, while the CD spectrum of the mostretained chromatographic fraction contained a positive CD band at thesame wavelength (FIG. 2B). These results indicate that each of thefractions contained one of the fenoterol enantiomers.

The sign of the lowest energy CD band can be used to assign the absoluteconfiguration to the separated fenoterol enantiomers, by applying theBrewster-Buta/Smith-Fontana sector rule for chiral benzylic derivatives(Brewster and Buta, J. Am. Chem. Soc., 88: 2233-2240, 1996). This sectorrule is used to predict the sign of the CD band related to the ¹L_(b)electronic transition of the benzylic compounds, with either hydroxyl oramine moieties and has been primarily applied to conformationally mobilearomatic compounds containing a single stereogenic center. In the caseof fenoterol, there are two stereogenic centers. However, it is believedthat the observed optical activity is mainly determined by thearylcarbinol moiety, because of the distance between the aromatic ringand the stereogenic center. The application of this rule permitted theassignment of the absolute configuration of (S,S) to the fenoterolenantiomer contained in the less retained fraction that showed anegative CD band at 280 nm, and the absolute configuration of (R,R) tothe fenoterol enantiomer contained in the most retained fraction thatshowed a positive CD band at 280 nm. This assignment was confirmed bythe independent synthesis of (S,S)-fenoterol and (R,R)-fenoterol.

These studies indicate that (R,R)-fenoterol and (S,S)-fenoterol can beseparated from (±)-fenoterol to a high degree of enantiomeric purity.

Example 3 Chromatographic Determination of the Binding of(R,R)-Fenoterol and (S,S)-Fenoterol to the Immobilized β2-AR

This example demonstrates that (R,R)-fenoterol is responsible for theβ2-AR binding of the clinically used drug (±)-fenoterol.

The preparation, characterization and application of a liquidchromatographic stationary phase containing immobilized membranesobtained from the β2-AR HEK-293 cell line have been previously reported(Beigi et al., Anal. Chem., 76: 7187-7193, 2004). For example, Beigi etal. (Anal. Chem., 76: 7187-7193, 2004) demonstrated that frontaldisplacement chromatography could be used to determine the dissociationconstants (K_(d)) for the binding of two β2-AR antagonists,(S)-propranolol and CGP 12177A, to the immobilized β2-AR. Zonaldisplacement chromatography using CGP 12177A as the marker liganddemonstrated that the immobilized β2-AR had retained itsenantioselectivity as the addition of (S)-propranolol to the mobilephase produced a greater displacement than the addition of(R)-propranolol (Id.). The addition of (±)-fenoterol to the mobile phasewas also shown to produce a conformation change in the immobilized β3-AR(Id.). Agonist-induced conformational changes of the β2-AR, as well asmost G-protein coupled receptors, from a resting to active state havebeen documented (Ghanoui et al., Proc. Natl. Acad. Sci. U.S.A., 98:5997-6002, 2001).

Presently, the immobilized β2-AR column was equilibrated with therunning buffer containing the [³H]-fenoterol, the marker ligand, beforethe initiation of the displacement studies. It was assumed that thebinding data calculated using frontal displacement chromatographyreflects the binding of (R,R)-fenoterol and (S,S)-fenoterol to theactive state of the receptor. In frontal chromatography, the initialflat portion of the chromatographic trace represents the binding of amarker ligand that is specific for the immobilized target, in this studythe β2-AR, as well as non-specific binding to other sites on theimmobilized membrane fragments. Saturation of specific binding sitesproduces a breakthrough front followed by a plateau representing theestablishment of a new equilibrium. The addition of a second compoundinto the mobile phase will produce a shift of the chromatographic traceto the left if the compound competes with the marker for binding to theβ2-AR. The relationship between the magnitude of this shift and theconcentration of the marker ligand can be used to calculate the bindingaffinity of the displacer for the target and the number of activebinding sites (Moaddel and Wainer, Anal. Chem. Acata, 546: 97-105,2006).

As shown in FIG. 3 (curve 1), the addition of [³H]-fenoterol to therunning buffer produced the expected frontal chromatography trace.Sequential addition of increasing concentrations of (R,R)-fenoterol tothe running buffer produced a corresponding shift in the chromatographictraces towards smaller retention volumes (FIG. 3, curves 2-4). Themagnitude and the shift and the corresponding concentrations of(R,R)-fenoterol were analyzed using Eqn 1 and the calculateddissociation constant, K_(d), was 472 nM and the amount of availablebinding sites [P] was 176 pmoles per column, r²=0.9999 (n=2).

The sequential addition of increasing concentrations of (S,S)-fenoterolto the running buffer produced no corresponding shift in thechromatographic traces toward shorter retention times. Thus,(S,S)-fenoterol had no significant affinity for the immobilized β2-AR.

In order to validate the chromatographic results, a standard membranebinding study was conducted using membranes obtained from the sameHEK-293 cell line used to create the immobilized β2-AR column. The datareflected the presence of a single binding site with (mean±SD)K_(d)=457±55 nM (n=4) for (R,R)-fenoterol and 109,000±10,400 nM (n=4)for (S,S)-fenoterol. These data indicate that frontal affinitychromatography on immobilized cellular membrane columns can be used todetermine the magnitude and enantioselectivity of ligand binding to thetarget receptor. Further, the results from the frontal affinitychromatography and ligand competition binding studies both demonstratethat (R,R)-fenoterol is responsible for the β2-AR binding of theclinically used drug (±)-fenoterol.

Example 4 Synthesis

General Procedures

All reactions were carried out using commercial grade reagents andsolvents. Tetrahydrofuran (THF) was dried by refluxing over sodium andbenzophenone. Dichloromethane was dried by refluxing over calciumhydride. Ultraviolet spectra were recorded on a Cary 50 Concentrationspectrophotometer. Optical rotations were done on a Rudolph ResearchAutopol IV. NMR Spectra were recorded on a Varian Mercury VMX 300-MHzspectrophotometer using tetramethylsilane as the internal standard. NMRmultiplicities were reported by using the following abbreviations: s,singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet;apt., apparent; and br, broad. Low resolution mass spectra were obtainedon a Finnigan LCQ^(Duo) LC MS/MS atmospheric pressure chemicalionization (API) quadrupole ion trap MS system equipped with bothelectrospray (ESI) and atmospheric pressure chemical ionization (APCI)probes. Analytical HPLC data was obtained using a Waters 2690Separations Module with PDA detection. Method (a): ThermoHypersil BDS100×4.6 mm C18 column, H₂O/CH₃CN/TFA. Method (b): Brownlee PhenylSpheri-5 100×4.6 mm, water/acetonitrile/TFA. Method (c): Vydac 150×4 mmC18 column, H₂O/isopropanol/TFA. Method (d): CHIRALPAK® AD-H 250×10 mm,95/5/0.05 CH₃CN/isopropanol/diethylamine. Merck silica gel (230-400mesh) was used for open column chromatography.

3′,5′-Dibenzyloxy-α-bromoacetophenone (46)

A solution of 2.4 mL (46 mmol) of Br₂ in 45 mL of CHCl₃ was addeddropwise over 1 h to a chilled, stirring solution of 9.66 g (29 mmol) of3′,5′-dibenzyloxyacetophenone (45) in 40 mL of CHCl₃. The resultingsolution was allowed to warm to room temperature over 1 hour with goodstirring, then poured into 100 mL of cold H₂O and transferred to aseparatory funnel where the CHCl₃ fraction was isolated, washed withbrine solution, dried (Na₂SO₄), filtered, and concentrated to 10.8 g.This material was applied to 500 g of silica gel, eluting with CHCl₃ toobtain 2.65 g (22%) of compound 46 as a white solid. ¹H NMR (CDCl₃) δ4.39 (s, 2H), 5.08 (s, 4H), 6.85 (t, 1H, J=2.1 Hz), 7.20 (d, 2H, J=2.4Hz), 7.31-7.44 (m, 10H).

General procedure for the Enantioselective Reduction of compound 46 to3′,5′-Dibenzyloxyphenylbromohydrins [(R)-8,(S)-8]

Under argon atmosphere, ˜0.06 mL (0.316 mmol, 10 mol %) of 5.0 Mboron-methyl sulfide complex (BH₃SCH₃) in diethyl ether was added in oneportion to a solution of 25 mg (0.16 mmol, 5 mol %) of the appropriatecis-1-amino-2-indanol in 3 mL of dry THF. This material under argon wasadded over 30 minutes to a solution of 1.3 g (3.16 mmol) of3′,5′-dibenzyloxy-α-bromoacetophenone in 20 mL of dry THF, while at thesame time adding in −0.05 mL pulses, 0.45 mL of 5.0 M boron-methylsulfide complex. The resulting solution was stirred under argon for 2hours, and then quenched with 3 mL of methanol, controlling gasevolution. Solvents were removed in vacuo and the resulting residuetaken up in 30 mL of CHCl₃ and washed with 25 mL of 0.2 M sulfuric acidfollowed by 20 mL of brine, then dried (Na₂SO₄), filtered, andevaporated.

(R)-(−)-3′,5′-Dibenzyloxyphenylbromohydrin [(R)-8]

Prepared with (1R,2S)-(+)-cis-1-amino-2-indanol as the enantioselectivereduction catalyst to give 1.02 g (78%) of (R)-8 as a fine white powder.¹H NMR (CDCl₃) δ 3.44 (dd, 1H, J=9.0, 10.5 Hz), 3.55 (dd, 1H, J=3.3,10.5 Hz), 4.79 (dd, 1H, J=3.3, 8.7 Hz), 4.97 (s, 4H), 6.51 (t, 1H, J=2.4Hz), 6.57 (d, 2H, J=1.8 Hz), 7.21-7.38 (m, 10H); [α]_(D)=−12.1° (c=1.0MeOH).

(S)-(+)-3′,5′-Dibenzyloxyphenylbromohydrin [(S)-8]

Prepared with (1S,2R)-(−)-cis-1-amino-2-indanol as the enantioselectivereduction catalyst to give 1.07 g (82%) of (S)-8 as a fine white powder.¹H NMR (CDCl₃) δ 3.43 (dd, 1H, J=9.0, 10.5 Hz), 3.55 (dd, 1H, J=3.3,10.5 Hz), 4.78 (dd, 1H, J=3.3, 8.7 Hz), 4.96 (s, 4H), 6.50 (t, 1H, J=2.4Hz), 6.57 (d, 2H, J=1.8 Hz), 7.21-7.39 (m, 10H); [α]_(D)=+11.8° (c=0.90MeOH).

4-Benzyloxyphenylacetone (34)

To 10.0 g (41.3 mmol) of 4-benzyloxyphenylacetic acid (31) was added, 20mL of acetic anhydride and 20 mL of pyridine, which was heated to refluxwith stirring under argon atmosphere for 6 hours. Solvents wereevaporated and residue dissolved in CHCl₃ (50 mL) and washed with 1NNaOH (2×50 mL). Dried organic layer (MgSO₄), filtered, and evaporated to11.8 g of an amber oil. Vacuum distillation at 0.1 mm Hg in an oil-bathset to 170° C. followed by silica gel chromatography eluting with 8/2CH₂Cl₂-hexanes gave 2.68 g (27%). ¹H NMR (CDCl₃) δ 2.14 (s, 3H), 3.63(s, 2H), 5.05 (s, 2H), 6.94 (d, 2H, J=8.7 Hz), 7.10 (d, 2H, J=8.7 Hz),7.26-7.47 (m, 5H).

Phenylacetone (35)

A solution of 20.4 g (0.15 mol) of phenylacetic acid, acetic anhydride(70 mL) and pyridine (70 mL) was heated to reflux with stiffing underargon atmosphere for 6 hours. Solvents were evaporated and residuedissolved in CHCl₃ (100 mL), washed with 1N NaOH (2×100 mL) and driedthe organic layer (MgSO₄), filtered, and evaporated to give 20.4 g.Vacuum distillation at 0.1 mm Hg in an oil bath set to 160° C., followedby silica gel chromatography eluting with 1/1 hexanes/CH₂Cl₂ gave 5.5 g(27%). ¹H (CDCl₃) δ 2.15 (s, 3H), 3.70 (s, 2H), 7.20-7.36 (m, 5H).

1-Naphthalen-1-yl-propan-2-one (36)

A solution of 37.2 g (20 mmol) of naphthoic acid (33), acetic anhydride(100 mL) and pyridine (100 mL) was heated to reflux with stirring underargon atmosphere for 6 hours. Evaporated solvents, dissolved residue inCHCl₃ (200 mL) and washed with 1N NaOH (2×150 mL), dried organic layer(MgSO₄), filtered, and evaporated to give 34.6 g. Distillation at 0.5 mmHg in an oil bath set to 170° C., followed silica gel chromatographyeluting with 1/1 hexanes/CH₂Cl₂ gave 9.7 g (26%). ¹H (CDCl₃) δ 2.11 (s,3H), 4.12 (s, 2H), 7.40-7.53 (m, 4H), 7.81 (d, 1H, J=8.4 Hz), 7.87-7.90(m, 2H).

General Procedure for Preparation of 2-benzylaminopropanes (37-39, 42,43)

To the appropriate ketone (1 eq) in CH₂Cl₂ (c=0.5 M), cooled to 0° C.was added glacial HOAc (1 eq), followed by benzylamine (1 eq) andNaBH(AcO)₃ (1.4 eq). The reaction mixture was warmed to room temperatureand stirred under argon for 20 hours. The reaction mixture was cooled(ice bath), 10% NaOH (5 eq) was added dropwise and then extracted intoCH₂Cl₂, washed with brine. The product was then dried (Na₂SO₄), filteredand evaporated.

1-(4-benzyloxy)-2-benzylaminopropane (37)

Prepared from 4-benzyloxyphenylacetone (34; 2.0 g, 8.3 mmol) to afford2.61 g (95%) as a tan solid. ¹H (CDCl₃) δ 1.10 (d, 3H, J=6.3 Hz),2.50-2.58 (m, 1H). 2.68-2.77 (m, 1H), 2.82-2.89 (m, 1H), 3.75 (dd, 2H,J=12 Hz, J=30 Hz), 5.05 (s, 2H), 6.90 (d, 2H, J=8.7 Hz), 7.04 (d, 2H,J=8.7 Hz), 7.17-7.42 (m, 10H); MS (APCI+) m/z (rel): 332 (100).

1-Phenyl-2-benzylaminopropane (38)

Prepared from phenylacetone (35; 5.5 g, 41 mmol) to afford 8.4 g (91%)as a tan solid. ¹H (CDCl₃) δ 1.09 (d, 3H, J=6.3 Hz), 2.61-2.81 (m, 2H),2.92 (m, 1H), 3.80 (dd, 2H), 7.14-7.30 (m, 10H); MS (APCI+) m/z (rel):226 (100).

1-(1′-Naphthyl)-2-benzylaminopropane (39)

Prepared from 1-naphthalen-1-yl-propan-2-one (36; 5.0 g, 27.1 mmol) toafford 7.0 g (94%) as a tan solid. ¹H (CDCl₃) δ1.14 (d, 3H, J=6.0 Hz),3.02-3.18 (m, 2H), 3.27 (m, 1H), 3.80 (dd, 2H, J=13.2, 43.8 Hz),7.13-7.23 (m, 5H), 7.31-7.48 (m, 4H), 7.73 (d, 1H, J=7.8 Hz), 7.83-7.86(m, 1H), 7.96-7.99 (m, 1H); MS (APCI+) m/z (rel): 276 (100).

1-(4′-Methoxyphenyl)-2-benzylaminopropane (42)

Prepared from 4-methoxyphenyl-acetone (40; 2.75 g, 13.1 mmol) to afford2.31 g (97%). ¹H (CDCl₃) δ1.10 (d, 3H, J=6.3 Hz), 2.56-2.75 (m, 2H),2.90 (m, 1H), 3.79 (s, 1H), 3.79 (m, 2H, J=13.2 Hz), 6.82 (d, 2H, J=8.7Hz), 7.07 (d, 2H, J=8.7 Hz), 7.18-7.32 (m, 5H); MS (APCI+) m/z (rel):256 (100).

1-(4′-nitrophenyl)-2-benzylaminopropane (43)

Prepared from 4-nitrophenyl-acetone (41; 4.95 g, 28 mmol) to afford 7.32g (98%) as an amber oil. ¹H (CDCl₃) δ 1.60 (d, 3H, J=6.3 Hz), 2.73-2.85(m, 1H), 3.00-3.12 (m, 2H), 3.86 (dd, 2H, J=26 Hz, J=60 Hz), 7.23-7.40(m, 5H), 7.30 (d, 2H, J=9.0 Hz), 8.14 (d, 2H, J=8.7 Hz). MS (APCI+) m/z(rel): 271 (100).

General procedure for Enantiomeric Separation of 2-benzylaminopropanes[(R)-10-14, (S)-10-14]

The appropriate racemic 2-benzylaminopropane (1 eq) was combined withthe appropriate optically active mandelic acid (1 eq) in methanol (c=0.5M) and refluxed until the solution homogenized, then cooled to RT. Thecrystals were filtered, collected, and recrystallized twice frommethanol (c=0.3 M) to afford the optically active2-benzylaminopropane.mandelic acid salt. The salts were converted to thefree amine for the purpose of collecting NMR and rotation data bypartitioning the mandelic acid salt between 10% K₂CO₃ and CHCl₃, dryingorganic extracts (Na₂SO₄) and evaporating.

(R)-(−)-1-(4′-benzyloxy)-2-benzylaminopropane [(R)-10]

A sample of 2.13 g (6.42 mmol) of 1-(4-benzyloxy)-2-benzylaminopropane(37) was reacted with 972 mg (6.42 mmol) of (R)-(−)-mandelic acid togive 295 mg (28% based on enantiomeric abundance) of the free amineafter workup. ¹H NMR (CDCl₃) δ 1.12 (d, 3H, J=6.3 Hz), 2.58-2.78 (m,2H), 2.82-2.91 (m, 1H), 3.75 (dd, 2H, J=12 Hz, J=30 Hz)), 5.07 (s, 2H),6.93 (d, 2H, J=8.7 Hz), 7.10 (d, 2H, J=8.7 Hz), 7.21-7.42 (m, 10H); MS(APCI+) m/z (rel): 332 (100); [α]_(D)=−19.1° (c=1.4, MeOH).

(S)-(+)-1-(4′-benzyloxy)-2-benzylaminopropane [(S)-10]

The washes from the separation of (R)-10 were concentrated andpartitioned between 50 mL of chloroform and 50 mL of 10% K₂CO₃ in water.Washed organics with brine, dried (Na₂SO₄), filtered and evaporated to1.70 g (5.1 mmol). The organics were brought to reflux with 782 mg (5.1mmol) of (S)-(+)-mandelic acid (as previously described) andcrystallized 3 times to obtain 670 mg of the (S)-amine.(S)-mandelic acidsalt. The (S)-amine.(S)-mandelic acid salt was triturated in ether thenpartitioned between 30 mL of chloroform and 20 mL of 10% K₂CO₃ in water.The organic partition was washed with brine, then dried (Na₂SO₄),filtered and evaporated to give 366 mg of the free amine (33% based onenantiomeric abundance). ¹H NMR (CDCl₃) δ 1.10 (d, 3H, J=6.3 Hz),2.58-2.78 (m, 2H), 2.82-2.91 (m, 1H), 3.76 (dd, 2H, J=12, 30 Hz), 5.06(s, 2H), 6.93 (d, 2H, J=8.7 Hz), 7.09 (d, 2H, J=8.7 Hz), 7.21-7.42 (m,10H); MS (APCI+) m/z (rel): 332 (100); [α]_(D)=+19.2° (c=1.5 MeOH).

(R)-(−)-1-(4′-Methoxyphenyl)-2-benzylaminopropane [(R)-11]

A sample of 3.02 g (11.8 mmol) of1-(4′-methoxyphenyl)-2-benzylaminopropane (42) was reacted with 1.8 g(11.8 mmol) (S)-(+)-mandelic acid to give 530 mg (35% based onenantiomeric abundance) of the free amine after workup. ¹H NMR (CDCl₃) δ1.10 (d, 3H, J=6.3 Hz), 2.57-2.76 (m, 2H), 2.88-2.94 (m, 1H), 3.79 (s,3H), 3.72-3.88 (m, 2H), 6.82 (d, 2H, J=8.7 Hz), 7.07 (d, 2H, J=8.4 Hz),7.15-7.31 (m, 5H); MS (APCI+) m/z (rel): 256 (100); [α]_(D)=−30.4°(c=1.25 MeOH).

(S)-(−)-1-(4′-Methoxyphenyl)-2-benzylaminopropane [(S)-11]

A sample of 3.36 g (13.2 mmol) of the racemate1-(4′-methoxyphenyl)-2-benzylaminopropane (42) was reacted with 2.0 g(13.2 mmol) of (R)-(−)-mandelic acid to give 740 mg (44% based onenantiomeric abundance) of the free amine after workup. ¹H NMR, (CDCl₃)δ 1.10 (d, 3H, J=6.2 Hz), 2.55-2.76 (m, 2H), 2.88-2.95 (m, 1H),3.73-3.88 (m, 2H), 3.79 (s, 3H), 6.80 (d, 2H, J=8.7 Hz), 7.08 (d, 2H,J=8.4 Hz), 7.15-7.30 (m, 5H); MS (APCI+) m/z (rel): 256 (100);[α]_(D)+30.5° (c=1.1 MeOH).

(R)-(−)-1-(4′-nitrophenyl)-2-benzylaminopropane [(R)-12]

A sample of 2.0 g (7.3 mmol) of 1-(4′-nitrophenyl)-2-benzylaminopropane(43) was reacted with 1.13 g (7.3 mmol) of (S)-(+)-mandelic acid to give486 mg (49% based on enantiomeric abundance) of the free amine afterworkup. ¹H NMR (CDCl₃) δ 1.60 (d, 3H, J=6.3 Hz), 2.73-2.85 (m, 1H),3.00-3.12 (m, 2H), 3.86 (dd, 2H, J=26 Hz, J=60 Hz), 7.23-7.40 (m, 5H),7.30 (d, 2H, J=9.0 Hz), 8.14 (d, 2H, J=8.7 Hz); MS (APCI+) m/z (rel):271 (100); [α]_(D)=−9.3° (c=1.0 MeOH).

(S)-(−)-1-(4′-nitrophenyl)-2-benzylaminopropane [(S)-12]

A sample of 2.0 g (7.3 mmol) of 1-(4′-nitrophenyl)-2-benzylaminopropane(43) was reacted with 1.13 g (7.3 mmol) of (R)-(−)-mandelic acid to give640 mg (65% based on enantiomeric abundance) of the free amine afterworkup. ¹H NMR (CDCl₃) δ 1.60 (d, 3H, J=6.3 Hz), 2.73-2.85 (m, 1H),3.00-3.12 (m, 2H), 3.86 (dd, 2H, J=26, 60 Hz), 7.23-7.40 (m, 5H), 7.30(d, 2H, J=9.0 Hz), 8.14 (d, 2H, J=8.7 Hz); MS (APCI+) m/z (rel): 271(100); [α]_(D)=+8.2° (c=1.0 MeOH).

(R)-(−)-1-Phenyl-2-benzylaminopropane [(R)-13]

A sample of 2.62 g (11.6 mmol) of 1-phenyl-2-benzylaminopropane (38) wasreacted with 1.77 g (11.6 mmol) of (S)-(+)-mandelic acid to give 747 mg(57% based on enantiomeric abundance) of the free amine after workup. ¹HNMR (CDCl₃) δ 1.13 (d, 3H, J=6.0 Hz), 2.62-2.84 (m, 2H), 2.92-2.99 (m,1H), 3.81 (dd, 2H, J=13.2, 34.5 Hz) 7.14-7.29 (m, 10H); MS (APCI+) m/z(rel): 226 (100); [α]_(D)=−24.5° (c=1.10 MeOH).

(S)-(−)-1-Phenyl-2-benzylaminopropane [(S)-13]

A sample of 5.0 g (22.2 mmol) of racemic 1-phenyl-2-benzylaminopropane(38) was reacted with 3.4 g (22.2 mmol) of (R)-(−)-mandelic acid to give2.15 g (86% based on enantiomeric abundance) of the free amine afterworkup. ¹H NMR (CDCl₃) δ 1.11 (d, 3H, J=6.0 Hz), 2.62-2.84 (m, 2H),2.92-2.99 (m, 1H), 3.81 (dd, 2H, J=13.2, 34.5 Hz), 7.14-7.29 (m, 5H); MS(APCI+) m/z (rel): 226 (100); [α]_(D)=+18.2° (c=0.85 MeOH).

(R)-(−)-1-(1′-Naphthyl)-2-benzylaminopropane [(R)-14]

The washes recovered from the separation of (S)-14 were concentrated andpartitioned between 40 mL of chloroform and 40 mL of 10% K₂CO₃ in water.The organic partition was washed with 20 mL of brine then dried (Na₂SO₄)to afford 1.16 g (4.2 mmol) of the free amine, which was reacted with640 mg (4.2 mmol) of (S)-(+)-mandelic acid. 588 mg (46% based onenantiomeric abundance) of the free amine was obtained. ¹H NMR (CDCl₃) δ1.07 (d, 3H, J=6.0 Hz), 3.02-3.18 (m, 2H), 3.27 (m, 1H), 3.74 (dd, 2H,J=13.2, 30.9 Hz), 7.13-7.23 (m, 5H), 7.31-7.48 (m, 4H), 7.73 (d, 1H,J=7.8 Hz), 7.83-7.86 (m, 1H), 7.96-7.99 (m, 1H); MS (APCI+) m/z (rel):276 (100); [α]_(D)=−5.8° (c=1.0 MeOH).

(S)-(−)-1-(1′-Naphthyl)-2-benzylaminopropane [(S)-14]

A sample of 2.6 g (9.4 mmol) of 1-(1′-naphthyl)-2-benzylaminopropane(39) was reacted with 1.44 g (9.4 mmol) of (R)-(−)-mandelic acid to give420 mg (21% based on enantiomeric abundance) of the free amine afterworkup. ¹H NMR (CDCl₃) δ 1.07 (d, 3H, J=6.0 Hz), 3.02-3.18 (m, 2H), 3.27(m, 1H), 3.74 (dd, 2H, J=13.2, 30.9 Hz), 7.13-7.23 (m, 5H), 7.31-7.48(m, 4H), 7.73 (d, 1H, J=7.8 Hz), 7.83-7.86 (m, 1H), 7.96-7.99 (m, 1H);MS (APCI+) m/z (rel): 276 (100); [α]_(D)=+6.3° (c=1.0 MeOH).

(R)-(−)-2-Benzylaminoheptane [(R)-15]

A sample of 0.65 mL (4.4 mmol) of (R)-(−)-2-aminoheptane (R-44) 0.44 mL(4.4 mmol) of benzaldehyde and 0.1 mL of HOAc were combined in 40 mL ofCH₂Cl₂ and then cooled to 0° C. To the reaction mixture was added 2.75mg (13 mmol) of sodium triacetoxyborohydride in one portion, which wasstirred under argon at room temperature for 28 hours. The reactionmixture was diluted with 30 mL of CH₂Cl₂, cooled in an ice bath and 80mL of 5% NaOH (in water) was added. Fractions were separated, organics(Na₂SO₄) dried and evaporated to 638 mg (71%) of (R)-15. ¹H NMR (CDCl₃)δ 0.88 (m, 3H), 1.08 (d, 3H J=6.6 Hz), 1.20-1.39 (m, 6H), 1.41-1.67 (m,2H), 3.62-3.77 (m, 1H), 3.75 (p, 2H, J=12 Hz), 7.17-7.41 (m, 5H); MS(APCI+) m/z (rel): 206 (100); [α]_(D)=+6.9° (c=1.0, MeOH).

(S)-(+)-2-Benzylaminoheptane [(S)-15]

A sample of 0.15 mL (1 mmol) of (S)-(+)-2-aminoheptane ((S)-44) 0.1 mL(1 mmol) of benzaldehyde and 0.1 mL of HOAc were combined in 10 mL ofCH₂Cl₂ and cooled to 0° C., then added 650 mg (3 mmol) of sodiumtriacetoxyborohydride in one portion. The reaction mixture was stirredunder argon at room temperature for 28 hours. The mixture was dilutedwith 10 mL of dichloromethane, cooled in an ice bath and 20 mL of 5%NaOH (in water) was added. Fractions were separated, organics were dried(Na₂SO₄) and evaporated to 154 mg (70%). ¹H NMR (CDCl₃) δ 0.88 (m, 3H),1.08 (d, 3H T=6.6 Hz), 1.19-1.37 (m, 6H), 1.41-1.67 (m, 2H), 3.62-3.77(m, 1H), 3.75 (p, 2H, J=12 Hz), 7.17-7.41 (m, 5H); MS (APCI+) m/z (rel):206 (100); [α]_(D)=+ 7.8° (c=1.0, MeOH).

Preparation of Fenoterol Analogs, Procedure A

To form the epoxide, the appropriate 3′,5′-dibenzyloxyphenylbromohydrin((R)-8) or (S)-8 (1 eq) was combined with K₂CO₃ (1.4 eq) in 1:1 THF/MeOH(c=0.3 M) and stirred for 2 hours under argon at room temperature. Thesolvent was removed and the residue partitioned between toluene and H₂O.The toluene fraction was isolated, dried (Na₂SO₄), filtered, andevaporated. The residue was dissolved with the appropriate freebenzylamine (R)- or (S)-10-15, 28 (0.95 eq) in a good amount of tolueneand evaporated again under high vacuum to remove trace H₂O. Theresulting colorless residue was heated to 120° C. under argon for 20hours, cooled and checked by ¹H NMR and mass spectrometry to confirmcoupling. The residue was dissolved in EtOH (c=0.07 M) with heat andtransferred to a Parr flask, where it was hydrogenated at 50 psi ofhydrogen over 10% (wt) Pd/C (10 mg cat/65 mg bromohydrin) for 24 hours.Complete debenzylation was confirmed by mass spectrometry. The mixturewas filtered through Celite, the filter cake rinsed with isopropanol,and the filtrate concentrated. The residue was dissolved in 1:1isopropanol/EtOH (c=0.2 M) and brought to reflux for 30 minutes with 0.5eq of fumaric acid. The reaction was cooled and the solvent removed. Thecrude material was purified by open column chromatograph or preparativechromatograph.

Column Separation of (R,R)-1 and (S,S)-1, Procedure B

A sample of 75 mg of fenoterol HBr was dissolved in 1.5 mL of 95/5/0.05CH₃CN/isopropanol/HNEt₂ and applied in 100 μL injections to a CHIRALPAK®AD-H 10×250 mm 5 μm semi-preparative column using a waters 2690Separations Module, PDA set to 280 nm. The eluting solvent was 95/5/0.05CH₃CN/isopropanol/HNEt₂, 5 mL/min. Retention times for (S,S) and (R,R)isomers were 4.8 min and 7.8 min, respectively.

(R,R)-(−)-Fenoterol [(R,R)-1]

Obtained according to Procedure B to give 40 mg collected afterevaporation. ¹H NMR (CD₃OD) δ 1.05 (d, 3H, J=6.3 Hz), 2.49 (q, 1H, J=6.9Hz), 2.62-2.74 (m, 2H), 2.80-2.91 (m, 2H), 4.55 (dd, 1H, J=5.1, J=3.3Hz), 6.16 (t, 1H, J=2.4 Hz), 6.27 (d, 2H, J=2.1 Hz), 6.68 (d, 2H, J=8.4Hz), 6.94 (d, 2H, J=8.4 Hz); ¹³C NMR (CD₃CN) δ 20.3, 43.2, 55.1, 55.2,72.4, 102.2, 105.4, 116.0, 131.3, 131.8, 147.4, 156.2, 159.0; UV (MeOH)λ_(max) 279 nm (∈ 2,760), 225 (12,900), 204 (32,600); MS (APCI+) m/z(rel): 304 (100, M+H); [α]_(D)=−29.0° (conc=0.2% MeOH); HPLC: (a) 0.1%diethylamine in H₂O, 0.50 mL/min, 254 nm, t_(R) 2.90 min, 99% pure; (d)t_(R) 7.8 min, >99% pure.

(S,S)-(+)-Fenoterol [(S,S)-1]

Obtained according to Procedure B to give 35 mg after evaporation. ¹HNMR (CD₃OD) δ 1.05 (d, 3H, J=6.6 Hz), 2.49 (q, 1H, J=7.2 Hz), 2.62-2.76(m, 2H), 2.80-2.94 (m, 2H), 4.55 (dd, 1H, J=4.8, J=3.3 Hz), 6.16 (t, 1H,J=2.1 Hz), 6.27 (d, 2H, J=2.4 Hz), 6.68 (d, 2H, J=8.4 Hz), 6.94 (d, 2H,J=8.4 Hz); ¹³C (CD₃CN) δ 20.3, 43.2, 55.0, 55.2, 72.4, 102.2, 105.4,116.0, 131.3, 131.8, 147.4, 156.2, 159.0; UV (MeOH) λhd max 279 nm (∈2,680), 224 (12,700), 204 (32,800); MS (APCI+) m/z (rel): 304 (100,M+H); [α]_(D)=+28.5° (conc=0.20% MeOH); HPLC: (a) 0.1% diethylamine inH₂O, 0.50 mL/min, 254 nm, t_(R) 2.72 min, >99% pure; (d) t_(R) 4.8min, >99% pure.

(R,S)-(−)-Fenoterol Fumarate [(R,S)-1]

Prepared from (R)-8 and (S)-10 according to Procedure A to give 168 mg(64%). ¹H NMR (CD₃OD) δ 1.22 (d, 3H, J=6.6 Hz), 2.64 (dd, 1H, J=9.9 Hz,J=13.2 Hz), 3.01-3.51 (m, 4H), 4.79 (dd, 1H, J=3.0 Hz, J=9.9 Hz), 6.23(t, 1H, J=2.4 Hz), 6.36 (d, 2H, J=2.1 Hz), 6.75 (s, 1H), 6.76 (d, 2H,J=8.4 Hz), 7.05 (d, 2H, J=8.1 Hz); ¹³C NMR (CD₃OD) δ 16.2, 39.1, 52.5,57.4, 70.4, 103.4, 105.3, 116.7, 127.8, 131.4, 135.2, 144.6, 157.7,160.0, 168.2; UV (MeOH) λ_(max) 278 nm (∈ 2,520), 205 (27,900); MS(ESI+) m/z (rel): 304 (100, M+H); [α]_(D)=−7.5° (conc=0.75% MeOH); HPLC:(a) 70/30/0.05. 1.00 mL/min, 282 nm, t_(R)1.35 min, >99% pure; (b)50/50/0.05. 1.0 ml, 0.50 mL/min, 254 nm, t_(R) 2.72 min, >99% pure; (d)t_(R) 4.8 min, 1.00 mL/min, 280 nm, t_(R) 2.10 min, 97.5% pure.

(S,R)-(+)-Fenoterol Fumarate [(S,R)-1]

Prepared from (S)-8 and (R)-10 according to Procedure A to give 104 mg(39%). ¹H NMR (CD₃OD) δ 1.22 (d, 3H, J=6.6 Hz), 2.64 (dd, 1H, J=9.9 Hz,J=13.5 Hz), 3.47-3.04 (m, 4H), 4.80 (dd, 1H, J=2.7, J=9.6 Hz), 6.23 (t,1H, J=2.4 Hz), 6.36 (d, 2H, J=2.1 Hz), 6.75 (s, 1H), 6.76 (d, 2H, J=8.4Hz), 7.05 (d, 2H, J=8.4 Hz); ¹³C NMR (CD₃OD) δ 16.2, 39.1, 52.5, 57.4,70.4, 103.4, 105.3, 116.7, 127.8, 131.4, 135.2, 144.6, 157.7, 159.9,168.2; UV (MeOH) λ_(max) 278 nm (∈ 2,640), 202 (36,600); MS (ESI+) m/z(rel): 304 (100, M+H), 413 (10); [α]_(D)=+6.4° (conc=0.50% MeOH); HPLC:(a) 70/30/0.05, 1.00 mL/min, 282 nm, t_(R)1.35 min, 95.9% pure; (b)50/50/0.05, 1.0 mL/min, 280 nm, t_(R) 2.06 min, 99% pure.

(R,R)-(−)-1-p-Methoxyphenyl-2-(β-3′,5′-dihydroxy-phenyl-β-oxy)ethylaminopropaneFumarate [(R,R)-2]

Prepared from (R)-8 and (R)-11 according to Procedure A to give 172 mg(38%). ¹H NMR (CD₃OD) δ 1.08 (d, 3H, J=6.3 Hz), 3.05-2.56 (m, 5H), 4.57(dd. 1H, J=8.4, 5.4 Hz), 6.16 (m, 1H), 6.26 (d, 2H, J=2.7 Hz), 6.81 (d,2H, J=8.7 Hz), 7.03 (d, 2H J=8.7 Hz); ¹³C NMR (CD₃OD) δ 18.8, 42.3,54.5, 55.6, 56.0, 72.6, 103.0, 105.4, 115.0, 131.1, 131.2, 131.3, 146.2,159.8, 159.9; UV (MeOH) λ_(max) 277 nm (∈ 3,590), 224 (17,700), 207(29,500); MS (ESI+) m/z (rel): 318 (100, M+H); [α]_(D)=−24.9° (c=0.8MeOH); HPLC: (a) 70/30/0.05, 1.0 mL/min, 282 nm, t_(R)1.54 min, 96.5%pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R)1.51 min, 95.9% pure.

(S,S)-(−)-1-p-Methoxyphenyl-2-(β-3′,5′-dihydroxy-phenyl-β-oxy)ethylaminopropaneFumarate [(S,S)-2]

Prepared from (S)-8 and (S)-11 according to Procedure A to give 318 mg(53%). ¹H NMR (CD₃OD) δ 1.15 (d, 3H, J=6.0 Hz), 2.58-3.22 (m, 5H), 3.77(s, 3H), 4.68 (dd, 1H, J=4.8, 8.4 Hz), 6.18 (t, 1H, J=2.1 Hz), 6.31 (d,2H, J=2.1 Hz), 2.23 (s, 0.5 H, fumarate), 6.84 (d, 2H, J=8.7 Hz), 7.10(d, 2H, J=9.0 Hz); ¹³C NMR (CD₃OD) δ 16.1, 39.9, 52.4, 54.5, 55.3, 70.4,101.9, 104.2, 114.0, 129.2, 130.1, 144.4, 158.7, 158.9; UV (MeOH)λ_(max) 277 nm (∈ 2,100), 224 (11.00), 205 (22,700); MS (ESI+) m/z(rel): 318 (100, M+H); [α]_(D)=+28.6° (c=0.95 MeOH); HPLC: (a)70/30/0.05, 1.0 mL/min, 282 nm, t_(R)1.67 min, 96.0% pure; (b)50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.51 min, 97.1% pure.

(R,S)-(−)-1-p-Methoxyphenyl-2-(β-3′,5′-dihydroxy-phenyl-β-oxy)ethylaminopropaneFumarate [(R,S)-2]

Prepared from (R)-8 and (S)-11 according to Procedure A to give 160 mg(38%). ¹H NMR (CD₃OD) δ 1.20 (d, 3H, J=6.6 Hz), 2.62-2.71 (m, 1H),2.98-3.20 (m, 3H), 3.30-3.42 (m, 2H), 4.73-4.81 (m, 1H), 6.21 (m, 2H),3.35 (m, 2H), 6.71 (s, 0.5H, fumarate), 6.56-6.89 (m, 2H), 7.11-7.19 (m,2H); ¹³C NMR (CD₃OD) δ 15.6, 38.5, 51.8, 54.5, 55.9, 69.7, 102.1, 104.1,114.1, 128.5, 130.2, 136.0, 143.8, 158.8, 159.1; UV (MeOH) λ_(max) 277nm (∈ 4,100), 224 (21,400), 203 (50,600); MS (ESI+) m/z (rel): 318 (100,M+H); [α]_(D)=−7.2° (c=1.5 MeOH); HPLC: (a) 70/30/0.05, 1.00 mL/min, 282nm, t_(R) 1.40 min, 99% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R)1.51 min, 96.1% pure.

(S,R)-(+)-1-p-Methoxyphenyl-2-(β-3′,5′-dihydroxyphenyl-β-oxy)ethylaminopropaneFumarate [(S,R)-2]

Prepared from (S)-8 and (R)-11 according to Procedure A to give 200 mg(51%). ¹H NMR (CD₃OD) δ 1.12 (d, 3H, J=6.0 Hz), 2.58-3.13 (m, 5H), 3.77(s, 3H), 4.62 (dd, 1H, J=3.6, 9.0 Hz), 6.15 (m, 1H), 6.30 (d, 2H, J=1.8Hz), 6.85 (d, 2H, J=8.7 Hz), 7.11 (d, 2H, J=8.7 Hz); ¹³C NMR (CD₃OD) δ18.2, 41.4, 54.1, 55.7, 56.5, 64.7, 103.0, 105.3, 115.1, 130.7, 131.3,145.9, 159.8, 160.0; UV (MeOH) λ_(max) 277 nm (∈ 3,150), 224 (3,310),205 (30,600); MS (ESI+) m/z (rel): 318 (100, M+H); [α]_(D)=+14.1°(c=0.95 MeOH); HPLC: (a) 70/30/0.05, 1.00 mL/min, 282 nm, t_(R) 1.42min, 97.7% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.52 min,97.8% pure.

(R,R)-(−)-5-{2-[2-(4-Aminophenyl)-1-methylethylamino]-1-hydroxyethyl}-1,3-benzenediolFumarate [(R,R)-3]

Prepared from (R)-8 and (R)-12 according to Procedure A to give 88 mg(42%). ¹H NMR (CD₃OD) δ 1.23 (m, 3H), 2.70-3.24 (m, 4H), 3.54 (m, 1H),4.84 (dd, 1H, J=3.3, 9.6 Hz), 6.23 (t, 1H, J=2.4 Hz), 6.38 (d, 2H, J=2.1Hz), 6.75 (s, 2H, fumarate), 7.35 (dd, 4H, J=8.1, 21.0 Hz); ¹³C (CD₃OD)δ 15.5, 39.6, 52.7, 56.6, 70.3, 103.4, 105.3, 123.8, 132.0, 132.1,135.2, 137.5, 144.7, 160.0, 168.1; UV (MeOH) λ_(max) 284 nm (∈ 1,520),206 (21,700); MS (ESI+) m/z (rel): 303 (100, M+H); [α]_(D)=−6.8°(conc=1.0% MeOH); HPLC: (a) 80/20/0.05, 0.70 mL/min, 276 nm, t_(R) 2.07min, 95.5% pure; (b) 50/50/0.05, 1.0 mL/min, 282 nm, t_(R) 2.60, 97.16%pure.

(S,S)-(+)-5-{2-[2-(4-Aminophenyl)-1-methylethylamino]-1-hydroxyethyl}-1,3-benzenediolFumarate [(S,S)-3]

Prepared from (S)-8 and (S)-12 according to Procedure A to give 56 mg(25%). ¹H NMR (CD₃OD) δ 1.23 (m, 3H), 2.62-3.27 (m, 4H), 3.55 (m, 1H),4.74-4.88 (m, 1H), 6.22 (t, 1H, J=1.8 Hz), 6.37 (d, 2H, J=2.4 Hz), 6.75(s, 2H, fumarate), 7.32 (dd, 4H, J=8.7, 25.8 Hz); ¹³C NMR (CD₃OD) δ15.5, 39.6, 52.5, 56.7, 70.7, 103.4, 105.3, 123.3, 131.8, 132.0, 135.2,136.9, 144.7, 160.0, 168.1; UV (MeOH) λ_(max) 284 nm (∈ 1,720), 207(28,400); MS (ESI+) m/z (rel): 303 (100, M+H), 329 (20); [α]_(D)=+11.1°(conc=0.50% MeOH); HPLC: (a) 80/20/0.05, 0.7 mL/min, 276 nm, t_(R) 2.01min, <99% pure; (b) 50/50/0.05, 1.0 mL/min, 282 nm, t_(R) 2.50 min,99.4% pure.

(R,S)-(−)-5-{2-[2-(4-Aminophenyl)-1-methylethylamino]-1-hydroxyethyl}-1,3-benzenediolFumarate [(R,S)-3]

Prepared from (R)-8 and (S)-12 according to Procedure A to give 72 mg(35%). ¹H NMR (CD₃OD) ∈ 1.23 (m, 3H), 2.73-3.24 (m, 4H), 3.51 (m, 1H),4.80 (dd, 1H, J=2.7, 9.6 Hz), 6.22 (t, 1H, J=2.1 Hz), 6.36 (d, 2H, J=2.4Hz), 6.75 (s, 2H, fumarate), 7.32 (dd, 4H, J=8.4, 25.2 Hz); ¹³C NMR(CD₃OD) δ 16.1, 39.12, 5.16, 56.9, 70.4, 103.4, 105.3, 123.4, 132.0,132.0, 135.2, 136.8, 144.6, 160, 168.10; UV (MeOH) λ_(max) 284 nm (∈1,620), 205 (27,200); MS (ESI+) m/z (rel): 303 (100, M+H), 134 (14);[α]_(D)=−7.5° (conc=0.50% MeOH); HPLC: (a) 80/20/0.05, 0.7 mL/min, 276nm, t_(R) 2.08 min, 95.0% pure; (b) 50/50/0.05, 1.0 mL/min, 282 nm,t_(R) 2.51 min, 97.4% pure.

(S,R)-(+)-5-{2-[2-(4-Aminophenyl)-1-methylethylamino]-1-hydroxyethyl}-1,3-benzenediolFumarate [(S,R)-3]

Prepared from (S)-8 and (R)-12 according to Procedure A to give 93 mg(42%). ¹H NMR (CD₃OD) δ 1.23 (d, 3H, J=6.3 Hz), 2.70-3.78 (m, 4H),3.42-3.62 (m, 1H), 4.80 (dd, 1H, J=3.0, 9.9 Hz), 6.22 (t, 1H, J=2.1 Hz),6.37 (d, 2H, J=2.1 Hz), 6.75 (s, 2H, fumarate), 7.33 (dd, 4H, J=8.4,26.7 Hz); ¹³C NMR (CD₃OD) δ 16.2, 39.1, 52.6, 56.9, 70.5, 103.4, 105.3,123.5, 132.1, 133.7, 135.2, 137.1, 144.7, 160.0, 168.1 UV (MeOH) λ_(max)284 nm (∈ 8,230), 207 (100,000); MS (ESI+) m/z (rel): 303 (100, M+H),134 (18); [α]_(D)=+11.4° (conc=0.50% MeOH); HPLC: (a) 70/30/0.05, 1.00mL/min, 280 nm, t_(R) 1.45 min, 99% pure; (b) 50/50/0.05, 1.0 mL/min,282 nm, t_(R) 2.63 min, 95.33% pure.

(R,R)-(+5-[1-Hydroxy-2-(1-methyl-2-phenylethylamino)ethyl]-1,3-benzenediolfumarate [(R,R)-4]

Prepared from (R)-8 and (R)-13 according to Procedure A to give 92 mg(26%). ¹H NMR (CD₃OD) δ 1.22 (m, 3H), 2.68-3.28 (m, 2H), 3.10-3.28 (m,2H), 3.53 (br-m, 1H), 4.75-4.80 (m, 1H), 6.24 (t, 1H, J=2.4 Hz), 6.38(d, 2H, J=2.1 Hz), 6.75 (s, 1H, fumarate), 7.22-7.33 (m, 5H); ¹³C NMR(CD₃OD) δ 15.5, 40.3, 56.9, 70.2, 103.4, 105.3, 128.3, 129.9, 130.3,135.2, 137.3, 144.6, 144.6, 159.9, 168.1; UV (MeOH) λ_(max) 277 nm (∈926), 204 (18,700); MS (APCI+) m/z 288 (100, M+H); [α]_(D)=−21.2°(conc=0.85% MeOH); HPLC: (a) 50/50/0.05, 1.00 mL/min, 282 nm; t_(R) 1.73min; 99% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.46 min, 97.5%pure.

(S,S)-(+)-5-[1-Hydroxy-2-(1-methyl-2-phenylethylamino)ethyl]-1,3-benzenediolfumarate [(S,S)-4]

Prepared from (S)-8 and (S)-13 according to Procedure A to give 184 mg(51%). ¹H NMR (CD₃OD) δ 1.21 (m, 3H), 2.70-3.13 (m, 2H), 3.15-3.23 (m,2H), 3.54 (br-m, 1H), 4.79-4.86 (m, 1H), 6.24 (t, 1H, J=2.1 Hz), 6.39(t, 2H, J=2.7 Hz), 6.76 (s, 1H, fumarate), 7.22-7.32 (m, 5H); ¹³C NMR(CD₃OD) δ 15.5, 40.3, 56.9, 70.2, 103.4, 105.3, 128.3, 129.9, 130.3,135.1, 137.3, 144.6, 144.6, 159.9, 168.1 UV (MeOH) λ_(max) 278 nm (∈1,510), 207 (26,600); MS (APCI+) m/z 288 (100, M+H); [α]_(D)=+19.3°(conc=0.90% MeOH); HPLC: (a) 50/50/0.05, 1.00 mL/min, 282 nm, t_(R) 1.49min; 98.4% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.35 min, 99%pure.

(R,S)-(−)-5-[1-Hydroxy-2-(1-methyl-2-phenylethylamino)ethyl]-1,3-benzenediolfumarate [(R,S)-4]

Prepared from (R)-8 and (S)-13 according to Procedure A to give 170 mg(45%). ¹H NMR (CD₃OD) δ 1.22 (m, 3H), 2.68-3.28 (m, 2H), 3.13-3.28 (m,2H), 3.53 (br-m, 1H), 4.76-4.80 (m, 1H), 6.23 (t, 1H, J=2.1 Hz), 6.37(t, 2H, J=3.0 Hz), 6.75 (s, 1H, fumarate), 7.24-7.37 (m, 5H); ¹³C NMR(CD₃OD) δ 16.3, 24.2, 39.8, 57.2, 70.5, 103.4, 105.3, 128.4, 130.0,130.4, 135.2, 137.4, 144.6, 160.1 UV (MeOH) λ_(max) 278 nm (∈ 1,110),205 (31,000); MS (APCI+) m/z (rel): 288 (100, M+H); [α]_(D)=−6.9°(conc=0.85% MeOH); HPLC: (a) 50/50/0.05, 1.00 mL/min, 282 nm, t_(R) 1.53min, 99% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.46 min, 98.5%pure.

(S,R)-(+)-5-[1-Hydroxy-2-(1-methyl-2-phenylethylamino)ethyl]-1,3-benzenediolfumarate [(S,R)-4]

Prepared from (S)-8 and (R)-13 according to Procedure A to give 212 mg(59%). ¹H NMR (CD₃OD) δ 1.22 (m, 3H), 2.72 (dd, 1H J=10.2, 13.2 Hz),3.11 (dd, 1H, J=10.2, 12.6 Hz), 3.18-3.27 (m, 2H), 3.48-3.61 (m, 1H),4.83 (dd, 1H, J=3.3, 9.9 Hz), 6.22 (t, 1H, J=2.4 Hz), 6.36 (d, 2H, J=2.4Hz), 6.75 (s, 1H, fumarate), 7.24-7.37 (m, 5H); ¹³C NMR (CD₃OD) δ 16.3,24.2, 39.8, 57.2, 70.5, 103.4, 105.3, 128.4, 130.0, 130.4, 135.2, 137.4,144.6, 160.1; UV (MeOH) λ_(max) 278 nm (∈ 1,680), 206 (35,500); MS(APCI+) m/z (rel): 288 (100, M+H), 270 (19, M-OH); [α]_(D)=+9.1°(conc=1.1%, MeOH); HPLC: (a) 50/50/0.05, 1.00 mL/min, 282 nm, t_(R) 1.51min, 99% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.43 min, 99%pure.

(R,R)-(−)-5-{1-hydroxy-2-[1-methyl-2-(1-naphthyl)ethylamino]ethyl}-1,3-benzenediolfumarate [(R,R)-5]

Prepared from (R)-8 and (R)-14 according to Procedure A to give 135 mg(46%). ¹H NMR (CD₃OD) δ 1.18-1.23 (m, 3H), 3.16-3.34 (m, 1H, 2H),3.69-3.74 (m, 2H), 4.78-4.80 (m, 1H), 6.23 (t, 1H, J=2.4 Hz), 6.38 (m,2H), 7.41-7.61 (m, 4H), 7.83 (d, 1H, J=7.5 Hz), 7.90 (d, 1H, J=7.8 Hz),8.10 (m, 1H); ¹³C NMR (CD₃OD) δ 16.2, 37.2, 54.5, 56.1, 70.3, 103.4,105.3, 124.3, 126.5, 127.0, 127.7, 129.3, 130.1, 133.2, 135.2, 135.6,144.7, 160.1, 168.2; UV (MeOH) λ_(max) 282 nm (∈ 5,860), 224 (50,900),208 (35,500); MS (APCI+) m/z (rel): 338 (100, M+H), 169 (15, fragment);[α]_(D)=−20.4 (conc=0.50% MeOH); HPLC: (a) 60/40/0.05, 1.00 mL/min, 282nm, t_(R) 2.08 min, 95.7% pure; (b) 50/50/0.05, 1.5 mL/min, 282 nm,t_(R) 2.20 min, 99% pure.

(S,S)-(+)-5-{1-hydroxy-2-[1-methyl-2-(1-naphthyl)ethylamino]ethyl}-1,3-benzenediolfumarate [(S,S)-5]

Prepared from (S)-8 and (S)-14 according to Procedure A to give 118 mg(40%) ¹H NMR (CD₃OD) δ 1.13-1.17 (m, 3H), 3.14-3.26 (m, 1H, 2H),3.61-3.76 (m, 2H), 4.44-4.75 (m, 1H), 6.18 (t, 1H, J=2.4 Hz), 6.33 (m,2H), 7.36-7.52 (m, 4H), 7.77 (dd, 1H, J=1.8, 7.5 Hz), 7.84 (d, 1H, J=8.1Hz), 8.04 (t, 1H, J=8.4 Hz); ¹³C NMR (CD₃OD) δ 16.1, 37.1, 54.4, 56.0,70.3, 103.3, 105.3, 124.3, 126.5, 127.0, 127.7, 129.2, 130.0, 133.2,135.2, 135.7, 144.5, 160.0, 168.1; UV (MeOH) λ_(max) 282 nm (∈ 6,210),223 (56,400), 208 (42,700); MS (APCI+) m/z (rel): 338 (100, M+H), 169(8, fragment); [α]_(D)=+20.0° (conc=1.1% MeOH); HPLC: (a) 60/40/0.05,1.00 mL/min, 282 nm, t_(R) 2.35 min, 98.9% pure; (b) 50/50/0.05, 1.5mL/min, 282 nm, t_(R) 2.26 min, 97.2% pure.

(R,S)-(−)-5-{1-hydroxy-2-[1-methyl-2-(1-naphthyl)ethylamino]ethyl}-1,3-benzenediolfumarate [(R,S)-5]

Prepared from (R)-8 and (S)-14 according to Procedure A to give 114 mg(39%). ¹H NMR (CD₃OD) δ 1.08-1.11 (m, 3H), 3.02-3.24 (m, 1H, 2H),3.54-3.68 (m, 2H), 4.45-4.75 (m, 1H), 6.11 (t, 1H, J=1.8 Hz), 6.26 (m,2H), 6.63 (s, 2H fumarate), 7.28-7.48 (m, 4H), 7.70 (d, 1H, J=7.5 Hz),7.77 (d, 1H, J=7.8 Hz), 7.97 (t, 1H, J=7.8 Hz); ¹³C NMR (CD₃OD) δ 16.0,37.1, 52.5, 56.0, 70.4, 103.4, 105.3, 124.4, 126.5, 127.0, 127.6, 129.2,130.1, 133.1, 135.2, 135.5, 144.7, 159.9, 168.2; UV (MeOH) λ_(max) 281nm (∈ 12,600), 224 (61,900), 204 (47,200); MS (APCI+) m/z (rel): 338(100, M+H), 190 (15, fragment); [α]_(D)=−11.3° (conc=0.85% MeOH); HPLC:(a) 60/40/0.05, 1.00 mL/min, 282 nm, t_(r) 2.30 min, 98.6% pure; (b)50/50/0.05, 1.5 mL/min, 282 nm, t_(R) 2.36 min, 99% pure.

(S,R)-(+)-5-{1-hydroxy-2-[1-methyl-2-(1-naphthyl)ethylamino]ethyl}-1,3-benzenediolfumarate [(S,R)-5]

Prepared from (S)-8 and (R)-14 according to Procedure A to give 123 mg(42%). ¹H NMR (CD₃OD) δ 1.18-1.22 (m, 3H), 3.10-3.28 (m, 1H, 2H),3.69-3.78 (m, 2H), 4.45-4.75 (m, 1H), 6.23 (t, 1H, J=2.1 Hz), 6.39 (m,2H), 6.73 (s, 2H fumarate), 7.39-7.59 (m, 4H), 7.80 (d, 1H, J=7.5 Hz),7.88 (d, 1H, J=7.8 Hz), 8.01 (t, 1H, J=9.0 Hz); ¹³C NMR (CD₃OD) δ 16.4,37.4, 52.5, 56.2, 70.6, 103.4, 105.3, 124.4, 126.5, 127.0, 129.3, 130.1,133.1, 133.4, 135.6, 136.3, 144.8, 160.0, 171.4; UV (MeOH) λ_(max) 282nm (∈ 7,740), 224 (70,900), 206 (55,800); MS (ESI+) m/z (rel): 338 (100,M+H); [α]_(D)=+15.5 (conc=1.0% MeOH)HPLC: (a) 60/40/0.05, 1.0 mL/min,282 nm, t_(R) 1.95, 95.7% pure; (b) 50/50/0.05, 1.5 mL/min, 282 nm,t_(R) 2.29 min, 95.7% pure.

(R,R)-(−)-5-[1-Hydroxy-2-(1-methylhexylamino)ethyl]-1,3-benzenediolFumarate [(R,R)-6]

Prepared from (R)-8 and (R)-15 according to Procedure A to give 45 mg(29%). ¹H NMR (CD₃OD) δ 0.920 (t, 3H, J=6.9 Hz), 1.30 (d, 3H, J=6.9 Hz),1.29-1.64 (m, 8H), 3.01-3.18 (m, 2H), 3.14-3.30 (m, 1H), 4.80 (dd, 1H,J=3.3, 9.6 Hz), 6.22 (t, 1H, J=2.1 Hz), 6.36 (d, 2H, J=2.4 Hz), 6.75 (s,1H, fumarate); ¹³C NMR (CD₃OD) δ 14.3, 16.0, 23.5, 26.2, 32.6, 34.2,52.1, 55.7, 70.2, 103.3, 105.3, 135.2, 144.7, 160.0, 168.0; UV (MeOH)λ_(max) 278 nm (E 931), 203 nm (20,100); MS (ESI+) m/z (rel): 268 (100,M+H); [α]_(D)=−8.8° (conc=1.1% MeOH); HPLC: (c) 70/30/0.1, 1.0 mL/min,276 nm, t_(R) 2.18 min, 96.6% pure; (b) 50/50/0.05, 1.0 mL/min, 279 nm,t_(R) 2.06 min, 98.9% pure.

(S,S)-(+)-5-[1-Hydroxy-2-(1-methylhexylamino)ethyl]-1,3-benzenediolFumarate [(S,S)-6]

Prepared from (S)-8 and (S)-15 according to Procedure A to give 96 mg(43%). ¹H NMR (CD₃OD) δ 0.923 (t, 3H, J=6.6 Hz); 1.31 (d, 3H, J=6.6 Hz),1.26-1.84 (m, 8H), 2.01-3.18 (m, 2H), 3.14-3.30 (m, 1H), 4.81 (dd, 1H,J=3.3, 9.6 Hz), 6.23 (t, 1H, J=2.4 Hz), 6.39 (d, 2H, J=2.1 Hz), 6.76 (s,1H, fumarate): ¹³C NMR (CD₃OD) δ 14.2, 16.0, 23.4, 26.3, 32.6, 34.1,52.1, 55.8, 70.2, 103.4, 105.3, 135.2, 144.7, 159.9, 168.2; UV (MeOH)λ_(max) 278 nm (∈ 1,340), 203 (28,800); MS (APCI+) m/z (rel): 268 (100,M+H); [α]_(D)=+10.8° (conc=0.50% MeOH); HPLC: (c) 70/30/0.1, 1.0 mL/min,276 nm, t_(R) 2.16 min, 97.0% pure; (b) 50/50/0.05, 1.0 mL/min, 279 nm,t_(R) 2.11 min, 99% pure.

(R,S)-(−)-5-[1-Hydroxy-2-(1-methylhexylamino)ethyl]-1,3-benzenediolFumarate [(R,S)-6]

Prepared from (R)-8 and (S)-15 according to Procedure A to give 83 mg(38%). ¹H NMR (CD₃OD) δ 0.924 (m, 3H); 1.32 (d, 3H, J=6.6 Hz), 1.26-1.84(m, 8H), 2.98-3.20 (m, 2H), 3.32-3.22 (m, 1H), 4.78 (dd, 1H, J=3.0, 9.9Hz), 6.23 (t, 1H, J=2.1 Hz), 6.37 (d, 2H, J=1.8 Hz), 6.76 (s, 1H,fumarate); ¹³C NMR (CD₃OD) δ 14.2, 16.4, 23.4, 26.2, 32.6, 33.5, 52.2,56.0, 70.4, 103.4, 105.3, 135.2, 144.7, 160.0, 168.1; UV (MeOH) λ_(max)276 nm (∈ 2,770), 203 (35,900); MS (APCI+) m/z (rel): 268 (100, M+H);[α]_(D)=−15.9° (conc=0.70% MeOH); HPLC: (c) 70/30/0.1, 1.0 mL/min, 276nm, t_(R) 2.16 min, 97.0% pure; (b) 50/50/0.05, 1.0 mL/min, 279 nm,t_(R) 2.07 min, 96.2% pure.

(S,R)-(+)-5-[1-Hydroxy-2-(1-methylhexylamino)ethyl]-1,3-benzenediolFumarate [(S,R)-6]

Prepared from (S)-8 and (R)-15 according to Procedure A to give 81 mg(38%). ¹H NMR (CD₃OD) δ 0.920 (t, 3H, J=6.3 Hz), 1.32 (d, 3H, J=6.9 Hz),1.30-1.77 (m, 8H), 2.99-3.17 (m, 2H), 3.23-3.26 (m, 1H), 4.76 (dd, 1H,J=3.0, 9.6 Hz), 6.22 (t, 1H, J=2.4 Hz), 6.36 (d, 2H, J=2.1 Hz), 6.75 (s,1H, fumarate); ¹³C NMR (CD₃OD) δ 14.2, 16.5, 23.5, 26.2, 32.6, 39.5,52.2, 56.0, 70.4, 103.4, 105.3, 135.2, 144.7, 160.0, 168.0; UV (MeOH)λ_(max) 278 nm (∈ 1,440), 204 (29,900); MS (APCI+) m/z (rel): 268 (100,M+H); [α]_(D)=+12.7° (conc=1.0% MeOH); HPLC: (c) 70/30/0.1, 1.0 mL/min,276 nm, t_(R) 2.16 min, 99% pure; (b) 50/50/0.05, 1.0 mL/min, 279 nm,t_(R) 2.02 min, 95.7% pure.

(R)-(−)-5-(1-Hydroxy-2-phenethylaminoethyl)-1,3-benzenediol fumarate[(R)-7]

Prepared from (R)-8 and 28 to give 37 mg (15%). ¹H NMR (CD₃OD) δ2.94-3.23 (m, 6H), 4.73 (dd, 1H, J=3.3, 9.9 Hz), 6.15 (t, 1H, J=2.4 Hz),6.29 (d, 2H, J=1.8 Hz), 7.19-7.28 (m, 5H), 6.69 (s, 1H); UV (MeOH)λ_(max) 278 nm (∈ 1,360), 205 (32,600); MS (APCI+) m/z (rel): 274 (100,M+H); [α]_(D)=−13.0° (conc=1.0% MeOH); HPLC: (a) 80/20/0.05, 1.00mL/min, 282 nm, t_(R) 1.47 min, 96.7% pure; (b) 50/50/0.05, 1.0 mL/min,272 nm, t_(R) 2.78 min, 95.1% pure.

(S)-(−)-5-(1-hydroxy-2-phenethylaminoethyl)-1,3-benzenediol fumarate[(S)-7]

Prepared from (S)-8 and 28 to give 51 mg (17%). ¹H NMR (CD₃OD) δ2.87-3.21 (m, 6H), 4.68 (dd, 1H, J=3.6, 9.9 Hz), 6.10 (t, 1H, J=2.4 Hz),6.24 (d, 2H, J=2.1 Hz), 6.63 (s, 1H), 7.12-7.21 (m, 5H); UV (MeOH)λ_(max) 278 nm (∈ 1,280), 204 (33,700); MS (APCI+) m/z (rel): 274 (100,M+H); [α]_(D)=+14.64° (conc=1.1% MeOH); HPLC: (a) 80/20/0.05, 1.00mL/min, 282 nm, t_(R) 1.47 min, 98.6% pure; (b) 50/50/0.05, 1.0 mL/min,272 nm, t_(R) 2.74 min, 98.8% pure.

(R,R)-(−)-ethylfenoterol

¹H NMR: (300 MHz, CD₃OD): δ 0.950 (t, 3H, J=7.5 Hz), 1.67 (m, 2H),2.83-3.18 (m, 4H), 3.33-3.40 (m, 1H), 3.37 (s, 4H), 4.82 (m, 1H), 6.24(d, 1H, J=2.1 Hz), 6.37 (d, 2H, J=1.8 Hz), 6.73 (s, 2H, fum), 6.76 (d,2H, J=8.4 Hz), 7.05 (d, 2H, J=8.7 Hz) ppm. CMR:

¹³C (75 MHz, CD₃OD): δ 9.43, 23.28, 36.56, 52.29, 62.16, 70.02, 103.4,105.3, 116.7, 127.8, 131.3, 136.5, 144.6, 157.6, 159.9, 172.3 ppm. UV:(Methanol), λ_(max) (∈): 206 nm (22,500), 223 (12,300), 278 (2,460). MS:(LCQ DUO ESI positive ion mass spectrum) M/z (rel): 318 (100, M+H). HPLC1: Column: Varian Sunfire C18 100×4.6; 70/30/0.1 water/acetonitrile/TFA;1.0 mL/min; Det: 278 nm; 2.76 min (fumarate, 6.99%), 3.57 min (90.11%);Purity: 97.1%. HPLC 2: Column: Chiralpak IA 250×10; 90/10/0.05acetonitrile/methanol/TFA; 2.0 mL/min; Det: 278 nm; 5.26 (RR isomer,92.37%), 7.11 min (fumarate, 5.02%); Purity 97.5%. Specific Rotation:[α]_(D)=−15.6 (free amine, 0.5% MeOH).

(R,S)-(−)-ethylfenoterol

¹H NMR: (300 MHz, CD₃OD): δ 0.972 (t, 3H, J=7.5 Hz), 1.70 (p, 2H, J=6.9Hz)), 2.86-3.22 (m, 4H), 3.32-3.37 (m, 1H), 3.34 (s, 4H), 4.82 (m, 1H),6.25 (t, 1H, J=2.1 Hz), 6.36 (d, 2H, J=1.8 Hz), 6.74 (s, 2H, fum), 6.77(d, 2H, J=8.4 Hz), 7.08 (d, 2H, J=8.7 Hz) ppm. CMR: ¹³C (75 MHz, CD₃OD):δ 9.820, 24.16, 36.48, 52.30, 62.32, 69.92, 103.3, 105.3, 116.8, 127.7,131.3, 136.1, 144.4, 157.6, 159.8, 171.3 ppm. UV: (Methanol), λ_(max)(∈): 204 nm (26,900), 224 (11,500), 278 (2,320). MS: (LCQ DUO ESIpositive ion mass spectrum) M/z (rel): 318 (100, M+H). HPLC 1: Column:Varian Sunfire C18 100×4.6; 70/30/0.1 water/acetonitrile/TFA; 1.0mL/min; Det: 278 nm; 2.79 min (fumarate, 3.34%), 3.56 min (96.11%);Purity: 99.5% HPLC 2: Column: Chiralpak IA 250×10; 90/10/0.05acetonitrile/methanol/TFA; 2.0 mL/min; Det: 278 nm; 5.88 (RS isomer,97.08%), 7.12 min (fumarate, 2.92%); Purity >99%. Specific Rotation:[α]_(D)=−7.2 (free amine, 0.5% MeOH).

C₂₂H₂₅NO₄.0.5C₄H₄O₄

¹H NMR: (300 MHz, CD₃OD): δ 1.22 (t, 3H, J=6.6 Hz), 3.09-3.21 (m, 3H),3.59-3.69 (m, 2H), 3.99 (s, 3H), 4.74-4.83 (m, 1H), 6.23 (t, 1H, J=2.4Hz), 6.37 (dd, 2H, J=2.4, 5.7 Hz), 6.74 (s, 1H), 6.86 (d, 1H, J=7.8 Hz),7.32 (d, 1H, J=7.8 Hz), 7.48 (t, 1H, J=6.9 Hz), 7.56 (t, 1H, J=6.9 Hz),8.02 (dd, 1H, J=8.4, 12.0 Hz), 8.27 (d, 1H, J=8.7 Hz) ppm. CMR: ¹³C (75MHz, CD₃OD): δ 15.78, 36.66, 52.39, 55.96, 70.20, 103.4, 104.5, 105.3,123.8, 124.3, 124.9, 126.2, 127.4, 128.1, 129.5, 133.8, 135.2, 144.6,156.6, 160.0, 168.3 ppm. UV: (Methanol), λ_(max) (∈): 298 nm (4,970),286 (9,920), 234 (22,600), 210 (42,500). MS: (LCQ DUO ESI positive ionmass spectrum) M/z (rel): 368 (100, M+H). Specific Rotation:[α]_(D)=−28.8 (Free Amine; 0.5% MeOH).

C₂₂H₂₅NO₄.0.5C₄H₄O₄

¹H NMR: (300 MHz, CD₃OD): δ 1.20 (t, 3H, J=6.6 Hz), 3.07-3.21 (m, 3H),3.52-3.75 (m, 2H), 3.97 (s, 3H), 4.69-4.83 (m, 1H), 6.24 (t, 1H, J=2.1Hz), 6.39 (dd, 2H, J=2.4, 5.4 Hz), 6.74 (s, 1H), 6.84 (d, 1H, J=7.8 Hz),7.31 (d, 1H, J=8.1 Hz), 7.48 (t, 1H, J=6.9 Hz), 7.56 (t, 1H, J=6.9 Hz),8.01 (dd, 1H, J=8.4, 13.5 Hz), 8.27 (d, 1H, J=7.8 Hz) ppm. CMR: ¹³C (75MHz, CD₃OD): δ 15.77, 36.64, 52.37, 55.94, 70.46, 103.4, 104.5, 105.3,123.8, 124.3, 124.9, 126.2, 127.4, 128.1, 129.4, 133.8, 135.5, 144.7,156.6, 160.0, 169.0 ppm. UV: (Methanol), λ_(max) (∈): 298 nm (5,430),286 (5,710), 233 (25,100), 210 (43,200). MS: (LCQ DUO ESI positive ionmass spectrum) M/z (rel): 368 (100, M+H). Specific Rotation:[α]_(D)=−15.8 (Free Amine; 0.5% MeOH).

A step in the synthesis of the 4 stereoisomers of 1-6 was the couplingof the epoxide formed from either (R)- or(S)-3′,5′-dibenzyloxyphenylbromohydrin with the (R)- or (S)-enantiomerof the appropriate benzyl-protected 2-amino-3-benzylpropane (1-5) or the(R)- or (S)-enantiomer of N-benzyl-2-aminoheptane (6), Scheme I.

The synthesis of (R)-7 and (S)-7 was accomplished using2-phenethylamine, Scheme II. This approach was similar to the onedeveloped by Trofast et al. (Chirality 3: 443-450, 1991) for thesynthesis of the stereoisomers of formoterol, compound 47, FIG. 5. Theresulting compounds were then deprotected by hydrogenation over Pd/C andpurified as the fumarate salts.

The chiral building blocks used in the syntheses were produced usingScheme III. The (R)- and (S)-3′,5′-dibenzyloxyphenyl-bromohydrinenantiomers were obtained by the enantiospecific reduction of3,5-dibenzyloxyα-bromoacetophenone using boron-methyl sulfide complex(BH₃SCH₃) and either (1R,2S)- or (1S,2R)-cis-1-amino-2-indanol. Therequired (R)- and (S)-2-benzylaminopropanes were prepared byenantioselective crystallization of the rac-2-benzylaminopropanes usingeither (R)- or (S)-mandelic acid as the counter ion.

Example 5 Binding Affinities of Exemplary Fenoterol Analogues for β1 andβ2 Adrenergic Receptors

This example demonstrates that fenoterol analogues have an equivalent ifnot greater binding affinity for β2-ARs than fenoterol.

Compounds were tested up to three times each to determine their bindingaffinities at the β₁- and β₂-ARs. Competition curves with standard andunknown compounds included at least six concentrations (in triplicate).For each compound, graphs were prepared containing individualcompetition curves obtained for that test compound. IC₅₀ values and Hillcoefficients were calculated using GraphPad Prism® software. K_(i)values were calculated using the Cheng-Prusoff transformation (BiochemPharmacol 22: 3099-3108, 1973). In each study, a standard compound wassimultaneously run on the 96-well plate. If the standard compound didnot have an IC₅₀ value close to the established average for thatcompound, the entire study was discarded and repeated again.

β₁-AR binding was done on rat cortical membrane following a previouslydescribed procedure (Beer et al., Biochem. Pharmacol. 37: 1145-1151,1988). In brief, male Sprague-Dawley rats weighing 250-350 g weredecapitated and their brains quickly removed. The cerebral cortices weredissected on ice, weighed and promptly transferred to a 50 ml test tubecontaining approximately 30 ml of 50 mM Tris-HCl, pH 7.8 (at roomtemperature). The tissues were homogenized with a polytron andcentrifuged at 20,000×g for 12 min at 4° C. The pellet was washed againin the same manner and resuspended at a concentration of 20 mg (originalwet wt) per 1 ml in the assay buffer (20 mM Tris-HCl, 10 mM MgCl₂, 1 mMEDTA, 0.1 mM ascorbic acid at pH 7.8). To block the β₂ sites present inthe cortical membrane preparation, 30 nM ICI 118-551 was also added tothe assay buffer. To wells containing 100 μl of the test drug and 100 μlof [³H]CGP-12177 (1.4 nM final concentration), 0.8 ml of tissuehomogenate was added. After 2 hours at 25° C., the incubation wasterminated by rapid filtration. Nonspecific binding was determined by 10μM propranolol.

HEK 293 cells stability transfected with cDNA encoding human β₂-AR(provided by Dr. Brian Kobilka, Stanford Medical Center, Palo Alto,Calif.) were grown in Dulbecco's Modified Eagle Medium (DMEM) containing10% fetal bovine serum (FBS), 0.05% penicillin-streptomycin, and 400μg/ml G418 as previously described (Pauwels et al., Biochem. Pharmacol.42: 1683-1689, 1991). The cells were scraped from the 150×25 mm platesand centrifuged at 500×g for 5 minutes. The pellet was homogenized in 50mM Tris-HCl, pH 7.7, with a Polytron, centrifuged at 27,000×g, andresuspended in the same buffer. The latter process was repeated, and thepellet was resuspended in 25 mM Tris-HCl containing 120 mM NaCl, 5.4 mMKCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, and 5 mM glucose, pH 7.4. The bindingassays contained 0.3 nM [³H]CGP-12177 in a volume of 1.0 ml. Nonspecificbinding was determined by 1 μM propranolol.

According to the above-described methods, binding affinities, expressedas K_(i) values, were determined using membranes obtained from a HEK 293cell line stably transfected with cDNA encoding human β₂-AR (Pauwels etal., Biochem. Pharmacol. 42: 1683-1689, 1991) with [³H]CGP-12177 as themarker ligand. The resulting IC₅₀ values and Hill coefficients werecalculated for each test compound using GraphPad Prism® software andK_(i) values were calculated using the Cheng-Prusoff transformation(Biochem Pharmacol 22: 3099-3108, 1973):K _(i)=IC₅₀/(1+L/K _(d))+Eqn. 1.Where: L is the concentration of [³H]CGP-12177 and K_(d) is the bindingaffinity of the [³H]CGP-12177. Each test compounds was assayed threetimes.

The relative binding affinities to the β₂-AR for the stereoisomers ofcompounds 1-4 and 6 were R,R>R,S>S,R≈S,S (FIG. 4; Table 1, below). Thisstereoselectivity is consistent with the previously reported potenciesof the formoterol stereoisomers (Trofast et al., Chiralty 3: 443-450,1991) and results from binding studies with the isoproterenol derivativePTFAM, compound 48, FIG. 5 (Eimerl et al., Biochem. Pharmacol. 36:3523-3527, 1987). With compound 5, no significant difference was foundbetween the K_(i) values of the R,R and R,S isomers, thus the order wasR,R=R,S>S,R>S,S. The K_(i) value for (R)-7 was greater than that of(S)-7, which is consistent with the established enantioselective bindingpreference for β₂-ARs with the R-configuration at the stereogenic centercontaining the β-OH moiety, c.f. (Eimerl et al., Biochem. Pharmacol. 36:3523-3527, 1987; Wieland et al., Proc. Natl. Acad. Sci. USA 93:9276-9281, 1996; Kikkawa et al., Mol. Pharmacol. 53: 128-134, 1998; andZuurmond et al., Mol. Pharmacol. 56: 909-916, 1999).

TABLE 1 The binding affinities to the β₂-AR of the compounds synthesizedin this study calculated as K_(i) ± SEM (nM), n = 3. Comparison of β₁-and β₂ adrenergic binding affinity of fenoterol isomers. Compound K_(i)β₁ K_(i) β₂ K_(i) β₁/K_(i) β₂ (R,R)-1 14750 ± 2510 345 ± 34 43 (R,S)-118910 ± 2367 3695 ± 246 5 (S,R)-1 >100,000 10330 ± 1406 NC(S,S)-1 >100,000 27749 ± 6816 NC (R,R)-2 21992 ± 3096 474 ± 35 46(R,S)-2 30747 ± 6499 1930 ± 135 16 (S,R)-2 33378 ± 9170 5269 ± 509 6(S,S)-2 >100,000 15881 ± 2723 NC (R,R)-3 24956 ± 2100 2934 ± 168 9(R,S)-3 31324 ± 3485 7937 ± 397 4 (S,R)-3 77491 ± 3583 23125 ± 2093 3(S,S)-3 31440 ± 1681 28624 ± 906  1 (R,R)-4 17218 ± 1270 1864 ± 175 9(R,S)-4 33047 ± 2779 6035 ± 434 4 (S,R)-4 >100,000 30773 ± 3259 NC(S,S)-4 >100,000 28749 ± 1811 NC (R,R)-5 3349 ± 125 241 ± 38 14 (R,S)-515791 ± 6269 341 ± 23 46 (S,R)-5 34715 ± 9092 1784 ± 148 19(S,S)-5 >100,000 2535 ± 209 NC (R,R)-6 10185 ± 499  9275 ± 902 1(R,S)-6 >100,000 31440 ± 1681 NC (S,R)-6 61295 ± 5821 >100,000 NC(S,S)-6 52609 ± 1434 56420 ± 5186 1 (R)-7 42466 ± 3466 10466 ± 1461 4(S)-7 52178 ± 3006 20562 ± 3721 3

When just the (R,R) isomers were compared, (R,R)-5 had the highestrelative affinity of the tested compounds, although the differencebetween (R,R)-5 and (R,R)-1 did not reach statistical significance,Table 1. The only other (R,R) stereoisomer with sub-micromolar affinitywas (R,R)-2, which had a significantly lower binding affinity than(R,R)-5, p=0.0051, and (R,R)-1, p=0.0291, although the mean K_(i) valuefor (R,R)-2 is only 23% greater than that of (R,R)-1. The minimal effectof transforming the p-OH moiety into a methyl ether is consistent withprevious data from Schirrmacher et al. (Bioorg. Med. Chem. Lett. 13:2687-92, 2003). In the previous study, rac-1 was converted into a[¹⁸F]-fluoroethoxy ether without significant loss of in vitro activityand it was concluded that, within the accuracy of the test measurements,the derivatization did not change the binding affinity of the rac-1 tothe β₂-AR.

Binding affinities, expressed as K_(i) values, for the β₁-AR weredetermined using rat cortical membranes with [³H]-CGP-12177 as themarker ligand (Beer et al., Biochem. Pharmacol. 37: 1145-1151, 1988.).The calculated K_(i) for (R,R)-5 was 3,349 nM and the binding affinitiesfor the all of the remaining test compounds were >10,000 nM, Table 1.Unlike the data from the β₂-AR binding studies, there was no clear trendwhich could be associated with the stereochemistry of the compounds.

The relative selectivity of the compounds for the β₂-AR and β₁-AR wasdetermined using the ratio K_(i)β₁/K_(i)β₂, Table 1. Of particularinterest were the ratios for the four compounds with sub-micromolaraffinity for the β₂-AR, (R,R)-1, (R,R)-2, (R,R)-5 and (R,S)-5, whichwere 46, 43, 14 and 46, respectively. The results for (R,R)-1 and(R,R)-2 are consistent with previously reported K_(i)β₁/K_(i)β₂ ratio of53 for the β₂-AR-selective agonist (R,R)-TA-2005, compound 49, FIG. 5.

The observed loss of β₂-AR selectivity for (R,R)-5 was unexpected as wasthe 3-fold increase in selectivity displayed by (R,S)-5 relative to(R,R)-5. Previous studies with the stereoisomers of 47 indicated thatboth the (R,R)- and (R,S)-isomers had a high degree of selectivity forthe β₂-AR, relative to the β₁-AR, with the selectivity of the(R,R)-isomer greater than that of the (R,S)-isomer (Trofast et al.,Chirality 3: 443-450, 1991). This is the case for compounds 1 and 2, butreversed for 5. It is also interesting to note that (S,R)-5 had asimilar selectivity (19-fold) and its affinity for the β₂-AR was only7-fold weaker than (R,R)-5, 1783 nM and 241 nM, respectively.

These studies demonstrate that (R,R)- or (R,S)-naphthyl fenoterolanalogues have a higher binding affinity for β2-ARs than any isoform offenoterol. The (R,R)-methyoxy fenoterol analogue has a similar K_(i) forthe β2-AR as (R,R)-fenoterol. Thus, such analogues are viable candidatesfor β2-AR agonists and can likely be used to treat disorders that arepresently treated with commercially available (±)-fenoterol.

Example 6 Comparative Molecular Field Analysis

This example illustrates the use of Comparative Molecular Field Analysis(CoMFA) to analyze the disclosed compounds.

The disclosed compounds were analyzed using Comparative Molecular FieldAnalysis, a 3D QSAR technique applicable to the analysis of the relativeactivities of stereoisomers and/or enantiomers at a selected target.

CoMFA was performed as implemented in SYBYL 7.2. (TRIPOS Inc., St.Louis, Mo.). Molecular models of all derivatives were prepared inHyperChem v. 6.03 (HyperCube Inc., Gainesville, Fla.) using ModelBuildprocedure to ensure the same conformation of the scaffold. Models wereextracted to SYBYL and the partial atomic charges (Gasteiger-Huckeltype) were calculated. Ligand models were aligned using as a commonsubstructure of the two asymmetric carbon atoms in the core of thefenoterol molecule (—C*—CH₂—NH—C*—CH₂—). Two types of molecular fields(steric and electrostatic) were sampled on the grid (2 Å spacing)lattice surrounding each structure. Distance-dependent dielectricconstant was used in electrostatic calculations and energetic cutoffs of30 kcal/mol for both the steric and the electrostatic energies were set.

The Partial Least Square correlation procedure applied for resultantdatabase extracted four statistically significant components and thefollowing validation parameters were obtained for the best solution:R²=0.920, F (4,21)=60.380, standard error of estimate=0.223,cross-validated (leave-one-out) R²=0.847. In general, electrostaticfields account for 48.1% of explained variance and steric fields accountfor 51.9%. The resulting 3D QSAR model shows good statisticalcorrelation with research data, R²=0.920 and F=60.380, and goodprediction power as indicated by the cross-validated R² value (Q²)=0.847and the standard error of prediction (SEP)=0.309, Table 2.

TABLE 2 The pK_(d) predicted by the CoMFA model. Derivative pKd MeasuredpKd Predicted (R,R)-1 6.46 5.84 (R,S)-1 5.43 5.48 (S,R)-1 4.99 5.02(S,S)-1 4.56 4.66 (R,R)-2 6.32 6.17 (R,S)-2 5.71 5.80 (S,R)-2 5.28 5.34(S,S)-2 4.80 4.99 (R,R)-3 5.53 5.57 (R,S)-3 5.10 5.21 (S,R)-3 4.64 4.75(S,S)-3 4.54 4.39 (R,R)-4 5.73 5.58 (R,S)-4 5.22 5.25 (S,R)-4 4.51 4.75(S,S)-4 4.54 4.43 (R,R)-5 6.62 6.72 (R,S)-5 6.47 6.36 (S,R)-5 5.75 5.90(S,S)-5 5.60 5.54 (R,R)-6 5.03 5.01 (R,S)-6 4.50 4.66 (S,R)-6 4.00 4.19(S,S)-6 4.25 3.84 (R)-7 4.98 5.33 (S)-7 4.69 4.51

In the first stage, the model was used to identify the regionsresponsible for the discrimination between the stereoisomers. The CoMFAprocedure produced several distinct asymmetric regions located in closeproximity of each chiral center. The first chiral center (carrying the βhydroxyl group) is surrounded by an electropositive region behind themolecule. An electropositive region can be associated with hydrogen bondformation and indicates favorable donor properties or unfavorableacceptor properties of the pseudoreceptor. In this case, the location ofthe electropositive field indicates that the orientation of the β-OHmoiety behind the plane of the model (the S configuration at the chiralcenter) would hinder H bond formation with the receptor. Theelectropositive region is closely associated with a steric unfavorableregion behind the first chiral center. This is an additional indicationthat the model demonstrates a preference for the β-hydroxyl group in theR configuration. The preference for the R configuration at this centeris consistent with previous models and research data, which demonstratedthat the R configuration is favored for functional activity at β-ARreceptors (c.f., Eimerl et al., Biochem. Pharmacol. 36: 3523-3527, 1987;Wieland et al., Proc. Natl. Acad. Sci. USA 93: 9276-9281, 1996; Kikkawaet al., Mol. Pharmacol. 53: 128-134, 1998; and Zuurmond et al., Mol.Pharmacol. 56: 909-916, 1999).

The CoMFA model also demonstrated the effect of the second chiralcenter. The preferred configuration can be derived from the bindingdata, where for compounds 1-4 and 6 the (R,R)-isomers had the higheraffinities relative to their respective (R,S)-isomers, while the K_(i)values for (R,R)-5 and (R,S)-5 were equivalent, Table 1. Thus, in thismodel, the more active isomers are those with the methyl moiety on thestereogenic center on the aminoalkyl portion of the molecules pointingout of the plane of the figure of the CoMFA model. This is depicted by asteric disfavoring region behind the second chiral center of themolecule, and indicates a preference for the R configuration at thissite.

In this study, only the aminoalkyl portion of the fenoterol molecule wasaltered and, therefore, the key CoMFA regions are associated with thisaspect of the molecule. In the resulting analysis, all four interactingregions were identified in the proximity of the aromatic moiety and allcan be used to generate hypotheses concerning the mode of binding actionof the studied derivatives.

In the model, the large electropositive region encompassing the areaclose to the —OH or OCH₃ substituents represents H-bond donor propertiesof the pseudoreceptor to these moieties. These interactions areresponsible for the relatively higher binding affinities of theO-derivatives, compounds 1 and 2, relative to compounds 3 and 4, in thelatter compound the p-amino substituent should be positively chargedunder the test conditions.

A large electronegative region and another electropositive region, bothlocated parallel on two sides of the aromatic system most likelyrepresent π-π or π-hydrogen bond interactions between the β₂-AR andelectron-rich aromatic moieties, such as the naphthyl ring. This isconsistent with the increased affinity of compounds 1, 2 and 5 relativeto the other compounds examined in this study. The role of thisinteraction is suggested by the observation that the K_(i) values for(R,R)-5 and (R,S)-5 were equivalent to (R,R)-1 and (R,R)-2, Table 1.

Two steric regions are located close to the electrostatic regions andone favors and the other disfavors bulkiness in the respective areas.This indicates that the binding of the aminoalkyl portions of themolecules are also sterically restricted.

The binding of agonists and antagonists to the β₂-AR has been studiedusing site-directed mutagenesis and molecular modeling techniques(Eimerl et al., Biochem. Pharmacol. 36: 3523-3527, 1987; Wieland et al.,Proc. Natl. Acad. Sci. USA 93: 9276-9281, 1996; Kikkawa et al., Mol.Pharmacol. 53: 128-134, 1998; Zuurmond et al., Mol. Pharmacol. 56:909-916, 1999; Kontoyianni et al., J. Med. Chem. 39: 4406-4420, 1996;Furse et al., J. Med. Chem. 46: 4450-4462, 2003; and Swaminath et al. J.Biol. Chem. 279: 686-691, 2004). There is general agreement that thebinding of the “catechol” portion of an agonist occurs within a bindingarea created by the transmembrane (TM) helices identified as TM3, TM5and TM6. The binding process is a sequential event that producesconformational changes leading to G-protein activation (Furse et al., J.Med. Chem. 46: 4450-4462, 2003). A key aspect in this process is theinteraction of the hydroxyl moiety on the chiral carbon of the agonistwith the Asn-293 residue in TM6, and for this interaction anR-configuration is preferable at the chiral carbon (Eimerl et al.,Biochem. Pharmacol. 36: 3523-3527, 1987; Kikkawa et al., Mol. Pharmacol.53: 128-134, 1998; and Swaminath et al. J. Biol. Chem. 279: 686-691,2004). Since the “catechol” portion of the fenoterol molecule was notaltered in this study, it follows that in the CoMFA model, anR-configuration at the first stereogenic center is preferred in moststable complexes.

The majority of the binding and functional studies of β₂-AR agonistshave been conducted with small N-alkyl substituents such as methyl,isopropyl and t-butyl, c.f. (Kontoyianni et al., J. Med. Chem. 39:4406-4420, 1996). However, while these compounds are active at theβ₂-AR, they are not subtype selective. This is illustrated by theK_(i)β₁/K_(i)β₂ ratios determined for compounds 49, 50 and 51 (FIG. 5)which were 53, 1.7 and 1.3, respectively (Kikkawa, et al. Mol.Pharmacol. 53: 128-134, 1998). The removal of the p-methoxyphenyl moietynot only reduced the selectivity, but also the affinities as therespective β₂K_(i) values were 12 nM, 170 nM and 6300 nM (Kikkawa, etal. Mol. Pharmacol. 53: 128-134, 1998).

The role that aminoalkyl substituents play in β₂-AR selectivity has beeninvestigated using site-directed mutagenesis and molecular modelingtechniques (Kikkawa, et al. Mol. Pharmacol. 53: 128-134, 1998; Furse etal., J. Med. Chem. 46: 4450-4462, 2003; and Swaminath et al. J. Biol.Chem. 279: 686-691, 2004). Using (R,R)-49 as the model ligand, Kikkawa,et al. determined that hydrogen bond formation between the p-methoxyoxygen on compound 49 and the hydroxyl group of tyrosine 308 (Y308)located in the extracellular end of TM7 was the source of the β₂-ARselectivity (Mol. Pharmacol. 53: 128-134, 1998).

Furse and Lybrand developed a de novo model of the β₂-AR andinvestigated molecular complexes of several ligands (agonist andantagonist) with this subtype (J. Med. Chem. 46: 4450-4462, 2003). Amongthe structures investigated, (R,R)-49 has the same aminoalkylsubstituent as the compound 2. Examination of the (R,R)-49/β₂-AR complexrevealed that the p-methoxy group oxygen of (R,R)-49 formed a hydrogenbond with the hydroxy group of Y308, which supports the model proposedby Kikkawa, et al. (Mol. Pharmacol. 53: 128-134, 1998). The distancebetween the two oxygen atoms bonded in the model was 3.22 Å. However,the methoxy moiety of the ligand was also located in close proximity tothree other polar residues, histidine 296 (H296) in TM6, tryptophan 109(W109) in TM3 and asparagine 312 (N312) in TM7, each of which caninteract with an aromatic group on the aminoalkyl portion of (R,R)-49.

In the Furse and Lybrand model, the distance between the oxygen atom ofthe ligand and the hydrogen atom of H296 was 5.88 Å and H296 wasproposed as an alternative hydrogen bond donor for interaction with themethoxy group of (R,R)-49. Since Y308 and H296 are found only in β₂-AR,the corresponding residues found in the β₁-AR are F359 and K347,respectively, the interaction with H296 and Y308 has been proposed asthe source of β₁/β₂ selectivity (Furse et al., J. Med. Chem. 46:4450-4462, 2003).

Since the previous studies of β₁/β₂ selectivity utilized (R,R)-49, thesubtype selectivity of the (R,R)-stereoisomers of the compoundssynthesized in our study were compared to the subtype selectivity of(R,R)-49. The data from this study suggest that hydrogen bond formationbetween Y308 and/or H296 and the oxygen atom on the p-substituent of theagonist is involved in β₂-AR selectivity. The interaction is possiblewith (R,R)-1 and (R,R)-2 and the K_(i)β₁/K_(i)β₂ ratios for thesecompounds are 43 and 46, respectively, which are comparable to theK_(i)β₁/K_(i)β₂ ratio of 53 determined for (R,R)-49. The K_(i)β₁/K_(i)β₂ratios for compounds 3, 4, 6 and 7 were <10 and reflect the fact thatthey do not have the ability to form hydrogen bonds with Y308 or H296.The hydrogen bonding interactions were also suggested by the CoMFA modelidentifying a large electropositive region surrounding the area close tothe —OH or —OCH₃ substituents, representing hydrogen-bond donorproperties of the pseudoreceptor.

The data from this study also suggest that an aromatic moiety on theaminoalkyl portion of the compound contributes to K_(i) and subtypeselectivity, even if the aromatic moiety is unable to form a hydrogenbond with the receptor. This is demonstrated by the comparison of theK_(i)β₂ values for the (R,R)-isomers of compounds 1-5 which were <3,000nM with K_(i)β₂ value of (R,R)-6 which was 9,000 nM and theK_(i)β₁/K_(i)β₂ ratios which were ≧9 for 1-5 while compound 6 displayedno subtype selectivity, Table 1. One possible mechanism to explain thedata is π-hydrogen bond formation. The cloud of π-electrons of aromaticrings can act as hydrogen bond acceptors, although it has been estimatedthat the interaction would be about half as strong as a normal hydrogenbond (Levitt and Perutz, J. Mol. Biol. 201: 751-754, 1998). The higheraffinity and subtype selectivity for (R,R)-5 relative to (R,R)-3 and(R,R)-4 or (R)-7 is consistent with the greater π electron distributionin the napthyl ring relative to the other aromatic rings.

The CoMFA model also identified a large electronegative region andanother electropositive region, both located parallel to the aromaticsystem, which are most likely associated with π-π or π-hydrogen bondinteractions between the β₂-AR and electron-rich aromatic moieties, suchas the naphthyl ring. Using the model developed by Furse and Lybrandwith (R,R)-49 as the interacting ligand, Y308, H296, W109 and N312 wereidentified as possible sources of π-π and/or π-hydrogen bondinteractions. In the β₂-AR model, the estimated distances between thep-methoxy moiety on (R,R)-49 and W109 and N312 were 4.80 Å and 3.45 Å,respectively. Since W109 and N312 are fully conserved in all β-ARsubtypes, the interactions suggested by the CoMFA model may representthe source of the increase affinities for (R,R)-1, (R,R)-2 and (R,R)-5,relative to the other (R,R)-isomers, but not the observed β₁/β₂selectivity.

The data from this study and the resulting CoMFA model indicate that thebinding process of the tested compounds with the β₂-AR includes theinteraction of the chiral center on the aminoalkyl portion of theagonist with a sterically restricted site on the receptor. The existenceof a sterically restricted site has been previously suggested from thedata obtained in the development of 3D models for agonist and antagonistcomplexes with the β₂-AR (Kobilka, Mol. Pharm. 65: 1060-1062, 2004). Forexample, (R,R)-49 and similar compounds with substituents larger than amethyl group at the stereogenic center on the aminoalkyl portion weresuggested to produce significant steric interactions that wouldunfavorably affect the ligand-receptor complexes.

The binding of an agonist to the β₂-AR has been described as a multistepinterrelated process, in which sequential interactions between theagonist and receptor produce corresponding conformational changes(Kobilka, Mol. Pharm. 65: 1060-1062, 2004). The CoMFA model reflects thefinal agonist/β₂-AR complex and, in order to discern the effect of thesteric restricted site, it is necessary to consider the effect thatinteraction with this site has on the outcome of the binding process. Adetailed description of the present CoMFA model is disclosed in Jozwiaket al. (J. Med. Chem., 50 (12): 2903-2915, 2007) which is herebyincorporated by reference in its entirety.

If one assumes that the interaction of the “catechol” portion of theagonist with the binding area created by TM3, TM5 and TM6 (the firstbinding area), then these interactions will fix the position of theaminoalkyl portion of the agonist relative to the steric restrictedsite, and perhaps even create this site. In the CoMFA model, the stericrestrictions at the site force the methyl moiety at the chiral center ofthe aminoalkyl portion to point out of the plane of the model.

Due to the free rotation about the N-atom, the configuration at thechiral center bearing the methyl moiety may likely not affect theability of the molecule to minimize the interaction with the stericrestricted site. However, in the minimum energy conformation, e.g., withthe methyl group pointing out of the plane of the CoMFA model, theorientation of the remaining segment of the aminoalkyl portion relativeto the second binding area would be affected by the stereochemistry.Indeed, R and S configurations would produce mirror image relationshipsto the second binding area. This situation is illustrated in FIG. 4where the catechol, first chiral center and the methyl moieties of(R,R)-5 and (R,S)-5 have been overlaid upon each other.

The studies elucidating the source of β₂-AR selectivity have primarilyutilized (R,R)-49 and one previous study of the effect of chirality onsubtype selectivity reported that (R,R)-47 had a higher β₂-ARselectivity than (R,S)-47 (Trofast et al., Chiralty 3: 443-450, 1991).Thus, the observed equivalent affinities and functional activities of(R,R)-5 and (R,S)-5 at the β₂-AR and the 3-fold increased β₂-ARselectivity of (R,S)-5 was an unexpected result. One possibleexplanation of these results is that the naphthyl moiety of (R,S)-5 doesnot interact with the site defined by Y308 and H296 and is directedtowards and binds to another site on the β₂-AR. This interaction alsoconveys or participates in subtype selectivity as well as increasedbinding affinity and agonist activity. Since the previous models ofβ₂-AR selectivity only employed (R,R)-isomers, it is possible that thissite has been overlooked.

Another explanation of the data is suggested by the “rockingtetrahedron” chiral recognition mechanism proposed by Sokolov andZefirov (Doklady Akademii Nauk SSSR 319: 1382-1383, 1991). In thisapproach to molecular chiral recognition, the enantiomeric ligands aresecured to a chiral selector by two binding interactions. Theinteractions must be non-equivalent and directional so that only oneorientation is possible. The tethered enantiomers still haveconformational mobility and the remaining moieties on the chiral centerwill sweep out overlapping but not identical steric volumes. Where andto what extent the chiral selector interacts with these steric volumes,determines the enantioselectivity of the process. If the chirality ofthe chiral selector places the interaction perpendicular to the plane ofthe ligand, no enantioselectivity is observed. As a deviation from theperpendicular increases, so does the enantioselectivity relative to theR or S configuration.

With (R,R)-5 and (R,S)-5, the interactions with the first binding areaand the steric restricted site of the CoMFA model are two non-equivalentand directional interactions that place the remaining constituents onthe second chiral center in the same, albeit mirror image, orientationrelative to the second binding area. As discussed above, theinteractions of the 1-napthyl moieties of compound 5 with Y308 and H296are believed to be the source of the observed β₂-AR selectivity. If the1-naphthyl rings sweep out overlapping but not identical steric volumes,then the observed K_(i)β₂ values and subtype selectivity indicate thefollowing: 1) the K_(i)β₂-AR values represent the sum total of theπ-hydrogen bond and π-π interactions between the 1-naphthyl moieties andY308 and H296, as well as additional non-β₂-AR specific interactionswith other residues such as W109 and N312; 2) the steric volume sweptout by (R,S)-5 increases the probability of interactions of Y308 andH296 with the π cloud of the naphtyl moiety relative to the (R,R)-5; and3) the steric volume swept out by (R,R)-5 increases the probability ofinteractions with non-β₂-AR specific sites relative to (R,S)-5.

The effect of the configuration at the second chiral center andconformational-based chiral selectivity is also illustrated by theaffinities and subtype selectivities of (R,R)-3, (R,S)-3 and (R)-7,Table 1. The inversion of the chirality at the second chiral carbon fromR to S, reduced the K_(i)β₂ value of the (R,S)-3/β₂-AR complex relativeto the (R,R)-3/β₂-AR complex by ˜3-fold while there was no significantdifference between their K₁β₁ values. The increased subtype selectivityobserved for (R,R)-3 relative to (R,S)-3, 9 versus 4, respectively,essentially reflects the differences in K_(i)β₂ values, which could be areflection of increased conformational energy required to bring thearomatic portion of the aminoalkyl chain into contact with theelectropositive and electronegative regions that comprise the secondbinding area or a decrease in the probability that this interactionwould occur.

The removal of the methyl moiety on the second chiral center, andthereby the chirality at this site ((R)-7), had a similar effect asinverting the chirality at this site from R to S. The K_(i)β₂ values for(R)-7 was 32% higher than (R,S)-3 and there was no difference in theβ₂-AR selectivity, Table 1. These results suggest that for compound 3,the primary effect of the R configuration at the second chiral site wasto direct the aminoalkyl chain towards the second binding area whichincreased the probability of interacting with this site and reduces theconformational energy required to achieve this interaction.

A difference between compounds 3 and 5 is the steric areas swept out bythe aromatic substituents. In the case of compound 3, the phenyl ringproduces a smaller, more linear area, while with compound 5, the1-naphthyl ring system produces a relatively larger and broader area.These differences can be used to guide the synthesis of additionalderivatives.

In an example, (R,R)-2 and (R,S)-5 are chosen as possible candidates forthe development of a new selective β₂-AR agonist. These compounds mayhave increased and extended systemic exposures relative to thecommercially available rac-1 due to changes in molecular hydrophobicity,metabolic profile and transporter interactions.

The present example provides a pharmacophore model which may be used asa structural guide for the design of new compounds with β₂-ARselectivity which can be tested for use in the treatment of a desiredcondition, including congestive heart failure.

Example 7 Pharmacokinetic Studies of (R,R)-Fenoterol,(R,R)-Methoxyfenoterol and (R,S)-Naphthylfenoterol

This example demonstrates the plasma concentrations of (R,R)-fenoterol,(R,R)-methoxyfenoterol and (R,S)-naphthylfenoterol administered as anintravenous (IV) bolus to male Sprague-Dawley rats.

(R,R)-fenoterol, (R,R)-methoxyfenoterol and (R,S)-naphthylfenoterol wereadministered to jungular vein cannulated (JVC) rats at a single dosageof 5 mg/ml intravenously (see Table 3). Dose calculations (mg/kg) werebased on the individual body weight measured on the day of treatment.Study duration for pharmacokinetic studies was 6 hours. Plasma sampleswere collected over six hours at the following nine timepoints: prior toadministration of the desired dose; 5.00-5.30 minutes after dose;15.00-16.30 minutes after dose; 30.00-33.00 minutes after dose; 60-65minutes after dose; 120-125 minutes after dose; 240-245 minutes afterdose; 300-305 minutes after dose; and 360-365 minutes after dose. Urinewas collected for 0-6 hours and 6 to 24 hours from 3 rats in eachtreatment group.

TABLE 3 Study conditions for measuring plasma concentrations of (R,R)-fenoterol, (R,R)-methoxyfenoterol and (R,S)-naphtylfenoterol. Dose DoseNo. of Rats level Concentration No. of for plasma Compound: (mg/kg):(mg/ml): Rats: analysis: (R,R)- 5 2.5 6 5 fenoterol (R,R)- 5 2.5 6 5methoxyfenoterol (R,S)- 5 2.5 6 2 naphtylfenoterol

Pharmacokinetic parameters for (R,R)-fenoterol, (R,R)-methoxyfenoteroland (R,S)-naphthylfenoterol after intravenous administration to rats (5mg/kg) were analyzed according to a two-compartment open model (seeTable 4). A drug that follows the pharmacokinetics of a two-compartmentmodel does not equilibrate rapidly throughout the body, as is assumedfor a one-compartment model. In the two-compartment model, the drugdistributes into two compartments, the central compartment and thetissue, or peripheral compartment. The central compartment representsthe blood, extracellular fluid, and highly perfused tissues. The drugdistributes rapidly and uniformly in the central compartment. A secondcompartment, known as the tissue or peripheral compartment, containstissues in which the drug equilibrates more slowly. Drug transferbetween the two compartments is assumed to take place by first-orderprocesses.

The following abbreviations are utilized in Table 4 below: alpha—macrorate constant associated with the distribution phase; beta—macro rateconstant associated with the elimination phase; A, B—zero time interceptassociated with the alpha phase and beta phase, respectively; AUC—areaunder the curve; T1/2 (K10)—half-life associated with the rate constantK10; K10—elimination rate—rate at which the drug leaves the system fromthe central compartment; K12—rate at which drug enters tissuecompartment from the central compartment; K21—rate at which drug enterscentral compartment from tissue compartment; V1—volume of distributionof the central compartment; V2—volume of distribution of the tissuecompartment; Vss—volume of distribution at steady state; andCl—clearance.

TABLE 4 Pharmacokinetic parameters for (R,R)-fenoterol, (R,R)-methoxyfenoterol and (R,S)-naphtylfenoterol after intravenousadministration to rats (5 mg/kg). (R,R)- (R,R)- (R,S)- fenoterolmethoxyfenoterol naphtylfenoterol (n = 2) (n = 5) (n = 5) Weight WeightWeight Parameter Units 306 ± 11 296 ± 8 297 ± 10 Two-compartment openmodel A μg/ml 1.6300 4.6437 4.0365 Alpha 1/min 0.0710 0.1982 0.1764 Bμg/ml 0.0577 0.3900 0.4372 Beta 1/min 0.0086 0.0054 0.0046 AUC min*μg/ml29.6861 96.1011 116.88 T_(1/2) (K10) min 12.19 13.23 18.11 K10 1/min0.0568 0.0524 0.0383 K12 1/min 0.0119 0.1309 0.1213 K21 1/min 0.01070.0203 0.0214 V1 ml 906.5 294.01 330.83 V2 ml 1005.20 1895.00 1872.92Vss ml 1911.70 2189.02 2203.75 Cl ml/min 51.54 15.40 12.66

Tables 5-7 illustrate the individual plasma concentrations of(R,R)-fenoterol, (R,R)-methoxyfenoterol and (R,S)-naphthylfenoterolafter IV administration to rats (5 mg/kg). The average concentration of(R,R)-fenoterol in plasma was dramatically lower (1.34 μg/ml) fiveminutes after IV administration to rats (5 mg/kg) compared to either theaverage concentration of (R,R)-methoxyfenoterol (2.12 μg/ml) or(R,S)-naphthylfenoterol (2.11 μg/ml).

TABLE 5 Individual plasma concentrations of (R,R)-fenoterol afterintravenous administration (5 mg/kg). Concentration (ug/ml) Time (min)Rat# 01 Rat #02 Average 5 1.34 1.34 15 0.36 0.36 30 0.17 0.50 0.34 600.05 0.05 0.05 120 0.03 0.01 0.02 240 0.0003 0.02 0.01 300 0.005 0.005360 0.08 0.03 0.06

TABLE 6 Individual plasma concentrations of (R,R)-methoxyfenoterol afterintravenous administration (5 mg/kg). Concentration (ug/ml) Time (min)Rat# 13 Rat# 14 Rat# 15 Rat# 16 Rat# 18 Average 5 1.94 2.14 2.51 1.892.12 15 0.48 0.54 0.62 0.67 0.56 0.58 30 0.31 0.40 0.46 0.48 0.38 0.4160 0.23 0.25 0.24 0.33 0.25 0.26 120 0.14 0.16 0.18 0.21 0.14 0.17 2400.09 0.12 0.17 0.14 0.08 0.12 300 0.06 0.07 0.07 0.09 0.07 0.07 360 0.050.06 0.05 0.08 0.04 0.06

TABLE 7 Individual plasma concentrations of (R,S)-naphthylfenoterolafter intravenous administration (5 mg/kg). Concentration (ug/ml) Time(min) Rat# 25 Rat# 26 Rat# 27 Rat# 28 Rat# 29 Average 5 2.52 2.16 1.642.10 2.11 15 0.85 0.78 0.50 0.60 0.68 30 0.49 0.54 0.34 0.33 0.43 600.36 0.42 0.37 0.29 0.24 0.34 120 0.25 0.29 0.26 0.22 0.18 0.24 240 0.110.11 0.13 0.13 0.11 0.12 300 0.10 0.11 0.12 0.11 0.10 0.11 360 0.08 0.080.11 0.10 0.09 0.09

The data demonstrate that the two derivatives, (R,R)-methoxyfenoteroland (R,S)-naphthylfenoterol, have a significantly higher systemicexposure (AUC) and longer clearance compared to (R,R)-fenoterol whichmay produce a longer acting drug. It is suggested that the longerclearance time may be the result of inhibiting glucuronidation.

Example 8 Inhibition of 1321N1 Astrocytoma Cell Growth by(R,R)-Fenoterol, Specific Fenoterol Analogues or Combination Thereof

This example demonstrates the ability of fenoterol and specificfenoterol analogues disclosed herein to inhibit 1321N1 astrocytoma cellgrowth in vitro and in vivo.

The possibility that selective β₂-AR (β₂-AR) agonists could affect thegrowth of gliomas and astrocytomas through the direct stimulation ofcAMP and/or associated pathways was determined. Previous studiesdemonstrated that β₂-AR are expressed in glioblastomas, eithermaintained as established cell lines or primary cultures derived fromhuman biopsies as well in the human-derived 1321N1 astrocytoma cellline. In the current study, the U87MG cell line was used as a negativecontrol as there was no detectable expression of the β₂-AR in thesecells and the sensitivity of the U87MG cell line to increased cAMPlevels has been previously established. The general structure andagonists used in this Example are provided below.

Name R₁ R₂ R₃ R₄ Isoproterenol —OH —H —CH₃ —CH₃ Fenoterol —H —OH —CH₃

Methoxyfenoterol —H —OH —CH₃

1-naphthylfenoterol —H —OH —CH₃

2-naphthylfenoterol —H —OH —CH₃

4-methoxy-1- naphthylfenoterol —H —OH —CH₃

Ethylfenoterol —H —OH —CH₂CH₃

4-methyloxy-ethylfenoterol —H —OH —CH₂CH₃

HEK cells transfected with human β₂-AR (HEK-β₂-AR, provided by Dr. BrianKobilka, Stanford Medical Center, Palo Alto, Calif.) were grown inDulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovineserum (FBS) and 0.05% penicillin-streptomycin with 400 μg/ml G418. The1321N1 astrocytoma cell line was obtained from European Collection ofCell Cultures (Sigma-Aldrich, St. Louis, Mo.) and the U87MG cells fromAmerican Type Culture Collection (Manassas, Va.). The cells werecultured in DMEM supplemented with 10% FBS. Drug treatments were carriedout when cells were 70-80% confluent.

Binding to cell membranes obtained from 1321N1 cells was conducted in a96-well format, as described previously (Jozwiak et al., J. Med. Chem.50: 2903-2915, 2007). In brief, the cells were scraped from the 150×25mm plates and centrifuged at 500×g for 5 minutes. The cell pellet waswashed twice, homogenized in Tris-HCl [50 mM, pH 7.7] and the crudemembranes were recovered by centrifugation at 27,000×g for 10 min. Thepellet was resuspended in Tris-HCl [25 mM, pH 7.4] containing 120 mMNaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, and 5 mM glucose. Thebinding assays contained 0.3 nM [³H]CGP-12177 in a volume of 1.0 mL, andsamples were conducted in triplicate. Nonspecific binding was determinedusing 1 μM propranolol. Total volume of incubation was 1.0 mL andsamples were incubated for 60 min at 25° C. The amount of protein in thebinding assay was 270 μg. The reaction was terminated by filtrationusing a Tomtec 96 harvester (Orange, Conn.) through glass fiber filters.Bound radioactivity was counted on a Pharmacia Biotech beta-plate liquidscintillation counter (Piscataway, N.J.) and expressed in counts perminute. IC₅₀ values were determined using at least six concentrations ofeach fenoterol analog, and calculated using Graphpad/Prism (ISI, SanDiego, Calif.). The K_(i) values were determined by the method of Chengand Prusoff.

β₂-AR mediated cAMP accumulation was determined as described previously(Jozwiak et al., J. Med. Chem. 50: 2903-2915, 2007). HEK-β₂-AR, 1321N1or U87MG cells were plated in 96-well plates. When the cells reachedconfluence, the medium was removed and each well rinsed with 0.1 mL ofKrebs-HEPES buffer (130 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.3 mMCaCl₂, 1.2 mM MgSO₄, 25 mM HEPES, and 10 mM glucose, pH 7.3). The plateswere preincubated for 10 minutes at room temperature with buffer alone;then test compound diluted in buffer was added to the wells forquadruplicate determinations. The plates were incubated for anadditional 10 minutes with the test compound. After incubation, themedium was removed and 0.1 mL of 0.5 M formic acid was added. After aminimum of 1 hour, the supernatant was removed and lyophilized. cAMP wasquantitated using the protein kinase binding assay of Gilman. The amountof protein per well was determined using the BCA protein determinationkit (Thermo Scientific Pierce, Rockford, Ill.) and was used to calculatethe amount of cAMP/mg/well.

To measure β₂-AR mediated inhibition of mitogenesis, HEK-β₂-AR, 1321N1or U87MG cells were seeded in a 96-well plate at approximately 5,000cells/well. After 48 hours, the wells were rinsed twice and the mediumwas replaced with fresh medium containing 10 μL of drug in sterilewater. After another 24 hours of incubation at 37° C., 0.25 μCi of[³H]-thymidine was added to each well. The cells were incubated for anadditional 2 hours at 37° C., at which point 10 μL of 10× trypsin wasadded, and the resuspended cells were harvested using a Tomtec 96harvester through glass fiber filters. DNA-associated radioactivity wasthe plate was counted as described above.

Cell cycle distribution was analyzed by flow cytometry. Briefly, cellswere trypsinized, washed with phosphate-buffered saline (PBS) and fixedwith 95% ethanol at −20° C. for 24 hours. Fixed cells were washed withPBS, treated with 0.05% RNase for 30 minutes at 37° C. and stained withpropidium iodide. The stained cells were analyzed using a FACScan laserflow cytometer (FACSCaliber, BD Biosciences).

Proteins were separated by 4-12% pre-cast gels (Invitrogen, Carlsbad,Calif.) using sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis and then electrophoretically transferred onto Hybond™-Pmembrane (Amersham Biosciences, Piscataway, N.J.). Blots were probedwith the following antibodies: Cyclin D1 (sc-246, mouse polyclonal IgG),Cyclin A (sc-596, rabbit polyclonal IgG), p27 (sc-528, Rabbit polyclonalIgG) and Actin (sc=1616, goat polyclonal IgG) all purchased from SantaCruz Biotechnology (Santa Cruz, Calif.) and p-Akt (Ser473, rabbitpolyclonal IgG) purchased from Cell Signaling Technology (Beverly,Mass.). The ECL Plus Western Blotting Detection System of AmershamBiosciences (Piscataway, N.J.) and the procedure recommended by themanufacturer were used for the detection of antigens. Protein bands werequantified by analyzing the images obtained using an Alphaimager™ S-3400(Alpha Innotech Corp., San Leandro, Calif.).

(R,R)-Fenoterol and the fenoterol analogues (Table 8) were synthesizedas described herein (also described in U.S. patent application Ser. No.12/376,945 filed Feb. 9, 2009, Jozwiak et al., J. Med. Chem., 50:2904-2915, 2007 and Jozwiak et al., Bioorg. Med. Chem., 18: 728-736,2010 each of which is incorporated by reference in its entirety).[³H]-CGP-12177 was purchased from PerkinElmer (Shelton, Conn.), DMEM waspurchased from Lonza Walkersville, Inc. (Walkersville, Md.), FBS waspurchased from Atlas Biologicals (Fort Collins, Colo.),Penicillin-Streptomycin and Geneticin (G418) were purchased fromInvitrogen (Carlsbad, Calif.), NaCl and CaCl₂ were purchased fromMallinckrodt (Phillipsburg N.J.) and (±)-propranolol, (R)-isoproterenol,forskolin, Tris-HCl, Trizma Base, PBS, KCl, MgSO₄, MgCl₂, D-(+)-glucose,KH₂PO₄ and HEPES were purchased from Sigma-Aldrich (St. Louis, Mo.).

Initial RT-PCR studies indicated that β₂-AR was expressed in 1321N1cells while β₁-AR was not. The expression of β₂-AR in 1321N1 cells wasconfirmed using displacement studies with [³H]CGP-12177 as the markerligand. Saturation studies determined that the binding affinity (K_(d)value) of CGP-12177 was 0.23 nM. The expression level of the β₂-AR(B_(max)) in the 1321N1 cellular membranes was 32 fmol/mg of protein.The β₂-AR selective antagonist ICI 118-551 bound to the membranes withhigh affinity (K_(d)=0.58 nM) and a Hill coefficient of ˜1.0 indicatinga single binding site. There was no observable binding of [³H]CGP-12177to the membranes obtained from U87MG cells indicating that the β₂-AR isnot expressed in this cell line.

The agonist-induced cAMP accumulation in 1321N1 cells was studied using(R)-isoproterenol and selected fenoterol derivatives. Each of theagonists with an R-configuration at the β-hydroxy carbon atom produced asignificant increase in cAMP production, FIG. 6. The calculatedEC_(50cAmp) value for (R)-isoproterenol was 16.5 nM). The EC_(50cAmp)values for the fenoterol analogs ranged from 13.5 nM to 88.24 nM, Table8. However, only (R,R)-fenoterol and (R,R)-methoxyfenoterol were fullagonists producing maximal cAMP accumulations of >100% relative to(R)-isoproterenol, while the cAMP accumulations produced by(R,R)-4-methoxy-1-naphthylfenoterol, (R,S)-4-methoxy-1-naphthylfenoteroland (S,S)-fenoterol were 35%, 53%, and 31%, respectively. The inducedcAMP accumulation produced by these compounds was blocked by theaddition of 1 μM propranolol and ICI 118-551 competitively antagonizedthe agonist activity of (R,R)-fenoterol, pA2=8.9 with a slope of−1.24±0.30.

TABLE 8 The activity of (R)-isoproterenol and fenoterol analogspresented as IC₅₀ values associated with the inhibition of[³H]-thymidine incorporation in 1321N1 cells, stimulation of cAMPaccumulation presented as EC_(50cAMP) determined in 1321N1 and HEK-β₂-ARcells. The IC₅₀ and EC_(50cAMP) values determined in 1321N1 cells arepresented as mean ± SD for n = 4. Stimulation Stimulation of cAMP ofcAMP Mitogenesis Accumulation Accumulation Inhibition 1321N1 HEK-β₂-ARCompound IC₅₀ (nM) EC_(50cAMP) (nM) EC_(50cAMP) (nM) (R)-isoproterenol0.05 ± 0.01 16.45 ± 6.14 0.2^(a) (R,R)-fenoterol 0.14 ± 0.07 15.91 ±2.04 0.3^(a) (R,S)-fenoterol 6.09 ± 1.93 4.7^(a) (S,R)-fenoterol 6.74 ±2.18 8.5 (S,S)-fenoterol 184.20 ± 26.10  1856.20 ± 925.76 580(R,R)-4-methoxyfenoterol 0.17 ± 0.02 13.50 ± 5.59 0.3^(a)(R,S)-4-methoxyfenoterol 2.01 ± 0.76 2.0^(a) (S,R)-4-methoxyfenoterol3.16 ± 0.71 7.2 (S,S)-4-methoxyfenoterol 337.20 ± 97.20  33.2(R,R)-1-naphthylfenoterol 1.57 ± 0.34 12.5^(a) (R,S)-1-naphthylfenoterol1.19 ± 0.38 2.7^(a) (S,R)-1-naphthylfenoterol 14.80 ± 5.59  66.7(S,S)-1-naphthylfenoterol 229.10 ± 57.40  29.7 (R,R)-ethylfenoterol 1.44± 0.27 2.8^(a) (R,S)-ethylfenoterol 17.88 ± 4.56  16.6^(a)(R,R)-2-naphthylfenoterol 1.91 ± 0.57 0.4^(a) (R,S)-2-naphthylfenoterol72.60 ± 29.31 7.6^(a) (R,R)-4-methoxy-1- 3.98 ± 0.28 68.97 ± 5.593.9^(a) naphthylfenoterol (R,S)-4-methoxy-1- 4.37 ± 0.70 88.24 ± 5.594.0^(a) naphthylfenoterol

Forskolin-induced increases in intracellular cAMP produced decreasedproliferation in 1321N1 cell line with a calculated IC₅₀ value of170.3±37.2 (FIG. 7A). Since the results of the studies with forskolindemonstrated that 1321N1 cells are sensitive to increases inintracellular cAMP concentrations, the effect of β₂-AR agonists oncellular proliferation in 1321N1 and U87MG cells was investigated. Inthis study, 1321N1 cells were incubated with β₂-AR agonists for 22hours, at which time [³H]-thymidine was added for an additional 2 hours,the cells were then harvested and [³H]-thymidine incorporation wasdetermined. Significant reductions in [³H]-thymidine incorporation wereobserved in a concentration-dependent manner for all of the compoundsused in the study (FIG. 7A). In particular, FIG. 7A shows that(R,R)-fenoterol (▪) is 1000 times more potent than both (S,S)-fenoterol(▾) and forskolin (●). The data was used to determine IC₅₀ valuesassociated with the inhibition of [³H]-thymidine incorporation and thevalues ranged from 0.05 nM observed with (R)-isoproterenol to 337.2 nMobserved with (S,S)-4-methoxyfenoterol, Table 8. The inhibitory effectof (R,R)-fenoterol was blocked by the addition of the β₂-AR antagonistpropranolol (1 μM) and competitively inhibited by ICI-118-551, pA2=8.9,slope −1.3±0.3, FIG. 7B.

The stimulation of cAMP accumulation in 1321N1 cells was examined for 6of the 19 compounds used in the mitogenesis inhibition studies and thecalculated EC_(50cAmp) and IC₅₀ values were compared. A log-logcorrelation of the data revealed an excellent correlation between thetwo values with an r²=0.93652. Although there were quantitativedifferences between the EC_(50cAmp) values of the 6 compounds determinedin 1321N1 cells and in HEK-β₂-AR cells, the correlation was repeatedusing the 19 EC_(50cAmp) values determined in the HEK-β₂-AR cells andthe 19 IC₅₀ values determined in the 1321N1 cells. A significantcorrelation was also observed between the two data sets, r²=0.72611. Theresults indicate that there is a significant relationship between cAMPstimulation in the HEK-β₂-AR cell line and the inhibition of[³H]-thymidine incorporation in 1321N1 cell line and that the HEK-β₂-ARcell line can be a useful screen for anti-mitogenesis activity.

The incubation of U87MG cells with β₂-AR agonists had no effect on[³H]-thymidine incorporation which is consistent with the lack ofexpression of the β₂-AR in this cell line. It is interesting to notethat β₂-AR agonists also had no effect on the proliferation of HEK-β₂-ARcells, although the compounds used in this study are highly active inthe stimulation of cAMP accumulation in these cells, Table 8. Thesestudies demonstrated that β2-AR agonists inhibit proliferation of 1321N1cells in vitro.

The effect of (R,R)-fenoterol on cell cycling in 1321N1 was determinedby treating the cells with various concentrations of (R,R)-fenoterol for20 hours followed by flow cytometric analysis. Untreated cells were usedas controls. (R,R)-fenoterol induced G₁ arrest with an associateddecrease in the proportion of cells in G₂ and S phase, as the proportionof cells in G₁ phase increased from 49.8% (controls) to 60.6-76% intreated cells, Table 9. The results also demonstrated that(R,R)-fenoterol arrested the cell cycle at doses as low as 0.1 nM, whichis consistent with the compound's ability to stimulate cAMP accumulationand inhibit [³H]-thymidine incorporation, Table 8. These results showthat activation of PKA by cAMP analogs induced cell growth arrest byblocking the cell cycle during the G₁ or G₂ phase.

TABLE 9 Effect of (R,R)-fenoterol on 1321N1 Cell Cycle Kinetics % PhaseDistribution (R,R)-fenoterol [nM] G1 G2 S Control 49.8 6.5 43.7 0.1 60.61.8 37.6 10.0 76.0 2.1 21.9 1000 74.7 0.7 24.6

The effect of (R,R)-fenoterol on selected molecular events associatedwith G₁ arrest in 1321N1 cells was examined using Western blot analysis.The data indicate that (R,R)-fenoterol significantly increased proteinlevels of the cyclin-dependent kinase inhibitor and p27 (FIG. 8A) andinhibited phosphorylation of Akt, at Ser-473, in a dose dependent mannerat nanomolar concentrations, FIG. 8B. At the same range ofconcentrations, (R,R)-fenoterol down-regulated the protein expression ofcyclin D1 and cyclin A, FIGS. 8C and 8D, but had only a modest effect onphosphorylation of mitogen-activated kinases ERK1/2, reachingsignificance at only a single concentration (FIG. 8E).

In the current study, treatment of 1321N1 cells with forskolin increasedthe basal intracellular concentration of cAMP from below 0.5 nmol/mgprotein to ˜4 nmol/mg protein and induced cell cycle arrest in _(G1)phase. Thus, the data indicate that the proliferation of 1321N1 cells isalso sensitive to changes in intracellular cAMP levels, that theselevels can be increased by treatment with β₂-AR agonists and that thiseffect is associated with the presence of functional β₂-AR in the 1321N1cells. The connection between β₂-AR stimulation and the inhibition ofmitogensis was supported by data from studies utilizing U87MG cells,which do not express functional β₂-AR. The treatment of U87MG cells withthe same series of β₂-AR agonists did not increase cAMP levels and hadno effect on [³H]-thymidine incorporation or cell proliferation. Inaddition, studies of the human-derived U118 glioma cell line indicatethat there is a low, but significant expression of β₂-AR in these cells.Treatment of U118 cells with (R,R)-fenoterol inhibited [³H]-thymidineincorporation with an IC₅₀ value that was 50-fold higher than the valuecalculated in the 1321N1 cells suggesting that the level of β₂-ARexpression affected the quantitative inhibitory activity of(R,R)-fenoterol.

The mechanism by which cAMP arrests cell growth has been characterizedin astrocytomas and other cell types. The data from these studiesindicate that cAMP can reduce cell growth by inhibiting the growthfactor-mediated cell proliferation signaling pathways such as ERK andPI3K (Cook and McCormick, Science 262(5136): 1069-1072, 1993; Sevetsonet al., J. Mol. Neurosci. 17(3): 1993; Kim et al., J. Biol. Chem.276(16): 12864-12870, 2001); Stork and Schmitt, Trends Cell Biol. 12(6):258-266, 2002), by elevating the levels of cell cycle inhibitor proteinsp21^(cip1) (Lee et al., 2000) and p27^(kip1) (van Oirschot et al., J.Biol. Chem. 276(36): 33854-33860, 2001) and/or by decreasing the levelof cyclin D1 protein (L'Allemain et al., Oncogene 14(16): 1981-1990,1997).

In this study, the data from studies utilizing (R,R)-fenoterol indicatedthat ERK1/2 activity, reported to be crucial for cyclin D1 induction wasonly modestly affected by (R,R)-fenoterol, although cyclin D1 wasnevertheless down regulated, as was Akt phosphorylation. Conversely, thecell cycle inhibitor p27 was up-regulated. It is quite likely that inthe 1321N1 cells the inhibition of cyclin D1 production by cAMP is atleast in part due to the inhibition of PI3K/Akt pathway. Since(R,R)-fenoterol inactivated Akt, it is possible that the increase inp27^(kip1) and decrease in cyclin D1 is a reflection of both directaction of cAMP on these proteins as well as an indirect action throughinactivation of Akt. In addition, the finding that (R,R)-fenoteroldecreased the level of cyclin A suggests that the fenoterol compoundscause growth inhibition through modulation of multiple phases of cellcycle.

All of the 19 compounds used in this Example had been characterizedpreviously (including herein) as full β₂-AR agonists in HEK-β₂-AR cells,with EC_(50cAmp) values ranging from 0.2 nM to 580 nM (see Jozwiak etal., J. Med. Chem., 50: 2904-2915, 2007 and Jozwiak et al., Bioorg. Med.Chem., 18: 728-736, 2010). The ability of 6 of the 19 compounds used inthis study to stimulate cAMP accumulation in 1321N12 cells wasdetermined and the results indicated that the compounds were weakeragonists in this cell line as compared to the HEK-β₂-AR cells, Table 8.However, although there were quantitative differences in the agonistactivities of the tested compounds, the calculated EC_(50cAmp) valuesfrom both the 1321N1 and HEK-β₂-AR cells were correlated to the observedinhibition of [³H]-thymidine incorporation. This observation isreflected in the observed activity of (S,S)-fenoterol, which is a weakpartial β₂-AR agonist in the 1321N1, FIG. 6, a full β₂-AR agonist (>100%accumulation) in HEK-β₂-AR cells and an effective inhibitor ofmitogensis, Table 8. The data obtained with (S,S)-fenoterol suggeststhat a very small increase in cAMP accumulation is sufficient to blockcell division in 1321N1 cells.

Using previously described techniques, the IC₅₀ data obtained in thisstudy and the related molecular structures of the fenoterol analogs wereused in a preliminary comparative molecular field analysis to generate astatistically valid model (R²=0.771; Q²=0.569; F=25.3, SEE=0.491).Unlike the CoMFA model obtained in the studies with the HEK-β₂-AR cellline, the results of this study suggest that the IC₅₀ values of thetested compounds are associated with the configurations at both of thefenoterol molecule's chiral centers of the compounds with the structureof the aromatic substituent on the aminoalkyl chain playing little or norole. The difference in the CoMFA models is reflected in the effects of(R,R)-ethylfenoterol and (S,S)-fenoterol in cardiomyocyte contractilityand the inhibition of [³H]-thymidine incorporation. Both of thesecompounds are essentially inactive in the rat cardiomyocytecontractility model with EC₅₀ values of 8,551 nM and 55,000 nM,respectively, while active inhibitors of mitogensis in the 1321N1 cellline with IC₅₀ values of 1.44 nM and 184.20 nM, respectively. Withoutbeing bound by a particular theory, it is contemplated that the vastdifferences between the activities of these compounds in the two testsystems may be the relative abundance of differing conformations of theβ₂-AR and the ability of the fenoterol analogs to stabilize or inducethese forms.

Additional studies were performed on a series of the N-alkyl derivativesof (R,R)-4-methoxy-fenoterol and characterized for activity in the1321N1 ([³H]-thymidine incorporation) and cardiomyocyte models (ratcardiomyocyte contractility). The data is as follows, all for the(R,R)-isomers: 4-methoxy-ethylfenoterol: IC₅₀ (1321N1)=14 nM; EC₅₀(cardio)>10,000 nM; and 4-methoxy-isopropylfenoterol: IC₅₀ (1321N1)=946nM; EC₅₀ (cardio)>10,000 nM.

The results of this study indicate that β₂-AR agonists inhibitedcellular replication in the 1321N1 cell line, that this effect wasblocked by the β₂-AR antagonist propranolol and that the β₂-AR agonistshad no effect on the growth of U87MG cells. The results also indicatethat the stereochemistries at the two chiral centers on the fenoterolmolecule play a key role in the level of inhibitory activity, and thatthe inhibition of mitogenesis in the 1321N1 cell line may stem from thebinding of the fenoterol derivatives to a conformation of the β₂-AR thatdiffers from the antagonist-bound conformation of the receptor exploredin the earlier studies.

Example 9 Brain and Plasma Analysis of [³H]-(R,R)-Methoxyfenoterol inMale Sprague-Dawley Rats

This example shows the brain-to-plasma distribution of[³H]-(R,R)-methoxyfenoterol after a single intravenous (IV)administration in male Sprague-Dawley rats.

Male Sprague-Dalwey rats (6 weeks of age, 206-220 grams) were randomlyassigned to treatment groups by a manual body weight stratificationprocedure. An overview of the research study design for evaluating thebrain-to-plasma distribution of [³H] (R,R)-methoxyfenoterol is providedin Table 10 below.

TABLE 10 Research Study design for Brain and Plasma Analysis of [³H]-(R,R)- methoxyfenoterol in Male Sprague-Dawley Rats. Dose Route No.Blood and (slow push Dose Dose of Brain Tissue over 30 Level Conc. MaleHarvest Group sec.) (mg/kg) (mg/ml) μCi/kg Rats Times (min)^(b) 1 IV 0^(a) 0 0 3 5 (con- trols) 2 IV 5 2.5 250 3 5 3 IV 5 2.5 250 3 15 4 IV5 2.5 250 3 30 5 IV 5 2.5 250 3 60 ^(a)Control group animals onlyreceived vehicle. ^(b)Animals were sacrificed at specified times inorder to collect blood and whole brain tissue.

The test article included (R,R)-methoxyfenoterol.0.5 fumarate (375.42)stored at room temperature and [³H]-(R,R)-methoxyfenoterol chloride(353.9) stored at 4° C. to −20° C. (specific activity 57 Ci/mmol);vehicle was sterile saline, 0.9% sodium chloride, USP. Dose formulationswere prepared on the day of the study. The amount of radioactivity inthe dose formulation was determined by liquid scintillation. The studylasted for an hour and radioactivity levels in plasma and brain tissuewere evaluated at the conclusion of the study. Approximately 5 mls ofwhole blood was collected. Blood samples were collected via cardiacpuncture, transferred into a tube containing K₃EDTA as the anticoagulantand kept on wet ice until processed to plasma. To generate plasma,samples were centrifuged within 15 minutes of collection at 2,500 RPMfor 15 minutes at room temperature. Plasma was divided equally intothree labeled cryovials and stored on ample dry ice until transferred toa freezer set at ≦−20° C. for storage until analysis. Duplicate aliquotswere weighed and then combined with scintillation cocktail. The [³H]radioactivity was determined on a liquid scintillation counter. Brainsamples were snap frozen in liquid nitrogen and stored at ≦−70° C. untilanalysis. Whole brain tissue was homogenized and the weight of thehomogenate was determined. Duplicate aliquots from the homogenate wereweighed, solubilized and then combined with scintillation cocktail. The[³H] radioactivity was determined on a liquid scintillation counter.

Male Sprague-Dawley rats were administered 5 mg/kg[³H]-(R,R)-methoxyfenoterol intravenously and the corresponding plasmaand brain levels were determined over 60 minutes. After 5 minutes,plasma levels where highest at 3.25±0.15 μg-equiv/ml and thensubsequently decreased by 62% to 1.24±0.43 μg-equiv/ml at 60 minutes(Table 11, FIG. 9). There was a similar, yet less dramatic trend with[³H]-(R,R)-methoxyfenoterol levels in the brain. The highest and lowestvalues were 0.92±0.10 and 0.70±0.37 μg-equiv/g, respectively. Acomparison of brain to plasma levels showed that after 15 minutes thatratio appeared to stabilize around 0.5. The actual percent dose found inthe brain was no more than 0.17% for any given animal.

TABLE 11 [³H]-(R,R)-methoxyfenoterol Levels and Dose Recovery in MaleRat Plasma and Brain Time Plasma (μg-equiv/ml) Brain (μg-equiv/g) Ratioof Brain/Plasma Percent of Dose in Brain (%) (min) Rat #^(a,b)Individual Mean SD Individual Mean SD Individual Mean SD Individual MeanSD 5 4 3.30 3.25 0.15 1.00 0.92 0.10 0.30 0.28 0.02 0.15 0.14 0.02 53.08 0.80 0.26 0.12 6 3.37 0.95 0.28 0.15 15 7 2.19 1.79 0.78 0.99 0.800.39 0.45 0.44 0.03 0.15 0.12 0.06 8 0.89 0.36 0.40 0.06 9 2.29 1.060.46 0.16 30 10 0.63 1.53 0.78 0.22 0.72 0.44 0.35 0.45 0.09 0.03 0.110.07 11 1.97 1.00 0.51 0.15 12 2.00 0.95 0.48 0.15 60 13 0.97 1.24 0.430.44 0.70 0.37 0.45 0.54 0.10 0.06 0.11 0.06 14 1.01 0.53 0.52 0.09 151.73 1.12 0.65 0.17 ^(a)Untreated rats (# 1-3) had no radioactivityabove background in plasma or brain. ^(b)Treated rats were administered5 mg/kg ³H-Methoxyfenoterol by intravenously.

These studies demonstrate that (R,R)-methoxyfenoterol is capable ofpassing through the blood brain barrier and that administration of suchcompound as well as likely other related fenoterol analogues andfenoterol by IV is an effective means of delivering these compounds tothe brain, such as to treat a brain tumor.

Example 10 Effect of (R,R)-Methoxyfenoterol Growth of 1321N1 XenograftImplanted in the Flank of SKID Mice

This example shows the ability of (R,R)-methoxyfenoterol to inhibit1321N1 tumor growth in vivo.

(R,R)-methoxyfenoterol was administered twice a day via IP (day 1-2, 0mg/kg/day; day 3-10, 30 mg/kg/day; and day 10-42, 50 mg/kg/day). FIG. 10illustrates the ability of (R,R)-methoxyfenoterol to inhibit growth of a1321N1 xenograft implanted in the flank of SKID mice.

These studies demonstrate that (R,R)-methoxyfenoterol inhibitsastrocytoma growth in vivo. Although these studies demonstrate that(R,R)-methoxyfenoterol is capable of inhibiting astrocytoma growth, oneof skill in the art will appreciate that they also provide support forusing other fenoterol analogues and fenoterol itself to treat anastrocytoma, but inhibiting astrocytoma growth, in additional subjects,including humans.

Example 11 Treatment of a Primary Brain Tumor

This example describes a method that can be used to treat a primarybrain tumor in a human subject by administration of a compositioncomprising fenoterol, a fenoterol analogue or a combination thereof at atherapeutically effective amount to reduce or inhibit on or more signsor symptoms associated with the primary brain tumor. Although particularmethods, dosages, and modes of administrations are provided, one skilledin the art will appreciate that variations can be made withoutsubstantially affecting the treatment.

A subject with an astrocytoma is selected based upon clinical symptoms.A biological sample is isolated from the subject and β2-AR expressiondetermined by Western Blot or histological studies. A positive resultindicates that the tumor may be treated by administration of fenoterol,a disclosed fenoterol analogue or a combination thereof. In oneparticular example, a tissue biopsy is obtained from a subject with aprimary brain tumor. β2-AR expression is determined in the sample. Thedetection of β2-ARs in the sample indicates that the primary brain tumorcan be treated by administration of a composition including(R,R)-4-methoxy-ethylfenoterol. (R,R)-4-methoxy-ethylfenoterol isadministered IP to the subject at a concentration of 30 mg/kg/day forthe first 10 days and 50 mg/kg/day for the remaining 32 days. Tumorgrowth is then assessed 7 days, 14 days, 21 days, 30 days and 42 daysfollowing treatment. In one example, the effectiveness of the treatmentis determined by imaging methods, including non-invasive,high-resolution modalities, such as computed tomography (CT) andespecially magnetic resonance imaging (MRI). For example, contrast agentuptake is monitored to determine the effectiveness of the treatment. Adecrease in permeability to the blood-brain barrier marked by an atleast 20% decrease in uptake of a contrast agent as compared toreference value or that measured prior to treatment indicates thetreatment is effective. Also, a twenty-percent reduction in tumor sizeas compared to tumor size prior to treatment is considered to be aneffective treatment.

Example 12 Use of Disclosed Compositions Including(R,R)-4-Methoxy-Ethylfenoterol and (S,R)-4-Methoxy-Ethylfenoterol as anAdjuvant Therapy

This example describes a method that can be used to reduce, prevent orretard tumor growth in a human subject that has been treated for amalignant astrocytoma.

A subject with an astrocytoma is selected based upon clinical symptoms.The primary form of treatment of the malignant astrocytoma is opensurgery. For subjects that are not surgical candidates, either radiationor chemotherapy is used as the initial treatment. Following the initialtreatment, a subject is administered a pharmaceutical compositioncontaining 3 parts (S,R)-4-methoxy-ethylfenoterol and 1 part(R,R)-4-methoxy-ethylfenoterol orally daily for an indefinite period oftime. The reoccurrence of tumor growth is monitored by imaging methods,including non-invasive, high-resolution modalities, such as CT and MRI.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method of treating glioblastoma, comprising:administering to a subject a therapeutically effective amount of acompound to reduce one or more symptoms associated with theglioblastoma, wherein the compound is an (R,R) stereoisomer and has theformula

wherein the compound is optically active.
 2. The method of claim 1,wherein the subject do not have a bleeding disorder prior toadministering a therapeutically effective amount of a compound.
 3. Themethod of claim 1, wherein treating the glioblastoma by reducing one ormore symptoms associated with the glioblastoma comprises inhibitingglioblastoma growth.
 4. The method of claim 1, further comprisingadministering an additional therapeutic agent, such as prior to,concurrent or subsequent to administering the compound of claim 1,wherein the additional therapeutic agent is a chemotherapeutic agent,such as carmustine, lomustine, procarbazine, streptozocin, or acombination thereof.
 5. The method of claim 1, wherein administering atherapeutically effective amount of a compound comprises administering atherapeutically effective amount of the compound with a pharmaceuticallyacceptable carrier.
 6. The method of claim 1, wherein the subject is ahuman.